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Raffi Hovasapian

Raffi Hovasapian

Enzymes VII: Km & Kcat

Slide Duration:

Table of Contents

I. Preliminaries on Aqueous Chemistry
Aqueous Solutions & Concentration

39m 57s

Intro
0:00
Aqueous Solutions and Concentration
0:46
Definition of Solution
1:28
Example: Sugar Dissolved in Water
2:19
Example: Salt Dissolved in Water
3:04
A Solute Does Not Have to Be a Solid
3:37
A Solvent Does Not Have to Be a Liquid
5:02
Covalent Compounds
6:55
Ionic Compounds
7:39
Example: Table Sugar
9:12
Example: MgCl₂
10:40
Expressing Concentration: Molarity
13:42
Example 1
14:47
Example 1: Question
14:50
Example 1: Solution
15:40
Another Way to Express Concentration
22:01
Example 2
24:00
Example 2: Question
24:01
Example 2: Solution
24:49
Some Other Ways of Expressing Concentration
27:52
Example 3
29:30
Example 3: Question
29:31
Example 3: Solution
31:02
Dilution & Osmotic Pressure

38m 53s

Intro
0:00
Dilution
0:45
Definition of Dilution
0:46
Example 1: Question
2:08
Example 1: Basic Dilution Equation
4:20
Example 1: Solution
5:31
Example 2: Alternative Approach
12:05
Osmotic Pressure
14:34
Colligative Properties
15:02
Recall: Covalent Compounds and Soluble Ionic Compounds
17:24
Properties of Pure Water
19:42
Addition of a Solute
21:56
Osmotic Pressure: Conceptual Example
24:00
Equation for Osmotic Pressure
29:30
Example of 'i'
31:38
Example 3
32:50
More on Osmosis

29m 1s

Intro
0:00
More on Osmosis
1:25
Osmotic Pressure
1:26
Example 1: Molar Mass of Protein
5:25
Definition, Equation, and Unit of Osmolarity
13:13
Example 2: Osmolarity
15:19
Isotonic, Hypertonic, and Hypotonic
20:20
Example 3
22:20
More on Isotonic, Hypertonic, and Hypotonic
26:14
Osmosis vs. Osmotic Pressure
27:56
Acids & Bases

39m 11s

Intro
0:00
Acids and Bases
1:16
Let's Begin With H₂O
1:17
P-Scale
4:22
Example 1
6:39
pH
9:43
Strong Acids
11:10
Strong Bases
13:52
Weak Acids & Bases Overview
14:32
Weak Acids
15:49
Example 2: Phosphoric Acid
19:30
Weak Bases
24:50
Weak Base Produces Hydroxide Indirectly
25:41
Example 3: Pyridine
29:07
Acid Form and Base Form
32:02
Acid Reaction
35:50
Base Reaction
36:27
Ka, Kb, and Kw
37:14
Titrations and Buffers

41m 33s

Intro
0:00
Titrations
0:27
Weak Acid
0:28
Rearranging the Ka Equation
1:45
Henderson-Hasselbalch Equation
3:52
Fundamental Reaction of Acids and Bases
5:36
The Idea Behind a Titration
6:27
Let's Look at an Acetic Acid Solution
8:44
Titration Curve
17:00
Acetate
23:57
Buffers
26:57
Introduction to Buffers
26:58
What is a Buffer?
29:40
Titration Curve & Buffer Region
31:44
How a Buffer Works: Adding OH⁻
34:44
How a Buffer Works: Adding H⁺
35:58
Phosphate Buffer System
38:02
Example Problems with Acids, Bases & Buffers

44m 19s

Intro
0:00
Example 1
1:21
Example 1: Properties of Glycine
1:22
Example 1: Part A
3:40
Example 1: Part B
4:40
Example 2
9:02
Example 2: Question
9:03
Example 2: Total Phosphate Concentration
12:23
Example 2: Final Solution
17:10
Example 3
19:34
Example 3: Question
19:35
Example 3: pH Before
22:18
Example 3: pH After
24:24
Example 3: New pH
27:54
Example 4
30:00
Example 4: Question
30:01
Example 4: Equilibria
32:52
Example 4: 1st Reaction
38:04
Example 4: 2nd Reaction
39:53
Example 4: Final Solution
41:33
Hydrolysis & Condensation Reactions

18m 45s

Intro
0:00
Hydrolysis and Condensation Reactions
0:50
Hydrolysis
0:51
Condensation
2:42
Example 1: Hydrolysis of Ethyl Acetate
4:52
Example 2: Condensation of Acetic Acid with Ethanol
8:42
Example 3
11:18
Example 4: Formation & Hydrolysis of a Peptide Bond Between the Amino Acids Alanine & Serine
14:56
II. Amino Acids & Proteins: Primary Structure
Amino Acids

38m 19s

Intro
0:00
Amino Acids
0:17
Proteins & Amino Acids
0:18
Difference Between Amino Acids
4:20
α-Carbon
7:08
Configuration in Biochemistry
10:43
L-Glyceraldehyde & Fischer Projection
12:32
D-Glyceraldehyde & Fischer Projection
15:31
Amino Acids in Biological Proteins are the L Enantiomer
16:50
L-Amino Acid
18:04
L-Amino Acids Correspond to S-Enantiomers in the RS System
20:10
Classification of Amino Acids
22:53
Amino Acids With Non-Polar R Groups
26:45
Glycine
27:00
Alanine
27:48
Valine
28:15
Leucine
28:58
Proline
31:08
Isoleucine
32:42
Methionine
33:43
Amino Acids With Aromatic R Groups
34:33
Phenylalanine
35:26
Tyrosine
36:02
Tryptophan
36:32
Amino Acids, Continued

27m 14s

Intro
0:00
Amino Acids With Positively Charged R Groups
0:16
Lysine
0:52
Arginine
1:55
Histidine
3:15
Amino Acids With Negatively Charged R Groups
6:28
Aspartate
6:58
Glutamate
8:11
Amino Acids With Uncharged, but Polar R Groups
8:50
Serine
8:51
Threonine
10:21
Cysteine
11:06
Asparagine
11:35
Glutamine
12:44
More on Amino Acids
14:18
Cysteine Dimerizes to Form Cystine
14:53
Tryptophan, Tyrosine, and Phenylalanine
19:07
Other Amino Acids
20:53
Other Amino Acids: Hydroxy Lysine
22:34
Other Amino Acids: r-Carboxy Glutamate
25:37
Acid/Base Behavior of Amino Acids

48m 28s

Intro
0:00
Acid/Base Behavior of Amino Acids
0:27
Acid/Base Behavior of Amino Acids
0:28
Let's Look at Alanine
1:57
Titration of Acidic Solution of Alanine with a Strong Base
2:51
Amphoteric Amino Acids
13:24
Zwitterion & Isoelectric Point
16:42
Some Amino Acids Have 3 Ionizable Groups
20:35
Example: Aspartate
24:44
Example: Tyrosine
28:50
Rule of Thumb
33:04
Basis for the Rule
35:59
Example: Describe the Degree of Protonation for Each Ionizable Group
38:46
Histidine is Special
44:58
Peptides & Proteins

45m 18s

Intro
0:00
Peptides and Proteins
0:15
Introduction to Peptides and Proteins
0:16
Formation of a Peptide Bond: The Bond Between 2 Amino Acids
1:44
Equilibrium
7:53
Example 1: Build the Following Tripeptide Ala-Tyr-Ile
9:48
Example 1: Shape Structure
15:43
Example 1: Line Structure
17:11
Peptides Bonds
20:08
Terms We'll Be Using Interchangeably
23:14
Biological Activity & Size of a Peptide
24:58
Multi-Subunit Proteins
30:08
Proteins and Prosthetic Groups
32:13
Carbonic Anhydrase
37:35
Primary, Secondary, Tertiary, and Quaternary Structure of Proteins
40:26
Amino Acid Sequencing of a Peptide Chain

42m 47s

Intro
0:00
Amino Acid Sequencing of a Peptide Chain
0:30
Amino Acid Sequence and Its Structure
0:31
Edman Degradation: Overview
2:57
Edman Degradation: Reaction - Part 1
4:58
Edman Degradation: Reaction - Part 2
10:28
Edman Degradation: Reaction - Part 3
13:51
Mechanism Step 1: PTC (Phenylthiocarbamyl) Formation
19:01
Mechanism Step 2: Ring Formation & Peptide Bond Cleavage
23:03
Example: Write Out the Edman Degradation for the Tripeptide Ala-Tyr-Ser
30:29
Step 1
30:30
Step 2
34:21
Step 3
36:56
Step 4
38:28
Step 5
39:24
Step 6
40:44
Sequencing Larger Peptides & Proteins

1h 2m 33s

Intro
0:00
Sequencing Larger Peptides and Proteins
0:28
Identifying the N-Terminal Amino Acids With the Reagent Fluorodinitrobenzene (FDNB)
0:29
Sequencing Longer Peptides & Proteins Overview
5:54
Breaking Peptide Bond: Proteases and Chemicals
8:16
Some Enzymes/Chemicals Used for Fragmentation: Trypsin
11:14
Some Enzymes/Chemicals Used for Fragmentation: Chymotrypsin
13:02
Some Enzymes/Chemicals Used for Fragmentation: Cyanogen Bromide
13:28
Some Enzymes/Chemicals Used for Fragmentation: Pepsin
13:44
Cleavage Location
14:04
Example: Chymotrypsin
16:44
Example: Pepsin
18:17
More on Sequencing Larger Peptides and Proteins
19:29
Breaking Disulfide Bonds: Performic Acid
26:08
Breaking Disulfide Bonds: Dithiothreitol Followed by Iodoacetate
31:04
Example: Sequencing Larger Peptides and Proteins
37:03
Part 1 - Breaking Disulfide Bonds, Hydrolysis and Separation
37:04
Part 2 - N-Terminal Identification
44:16
Part 3 - Sequencing Using Pepsin
46:43
Part 4 - Sequencing Using Cyanogen Bromide
52:02
Part 5 - Final Sequence
56:48
Peptide Synthesis (Merrifield Process)

49m 12s

Intro
0:00
Peptide Synthesis (Merrifield Process)
0:31
Introduction to Synthesizing Peptides
0:32
Merrifield Peptide Synthesis: General Scheme
3:03
So What Do We Do?
6:07
Synthesis of Protein in the Body Vs. The Merrifield Process
7:40
Example: Synthesis of Ala-Gly-Ser
9:21
Synthesis of Ala-Gly-Ser: Reactions Overview
11:41
Synthesis of Ala-Gly-Ser: Reaction 1
19:34
Synthesis of Ala-Gly-Ser: Reaction 2
24:34
Synthesis of Ala-Gly-Ser: Reaction 3
27:34
Synthesis of Ala-Gly-Ser: Reaction 4 & 4a
28:48
Synthesis of Ala-Gly-Ser: Reaction 5
33:38
Synthesis of Ala-Gly-Ser: Reaction 6
36:45
Synthesis of Ala-Gly-Ser: Reaction 7 & 7a
37:44
Synthesis of Ala-Gly-Ser: Reaction 8
39:47
Synthesis of Ala-Gly-Ser: Reaction 9 & 10
43:23
Chromatography: Eluent, Stationary Phase, and Eluate
45:55
More Examples with Amino Acids & Peptides

54m 31s

Intro
0:00
Example 1
0:22
Data
0:23
Part A: What is the pI of Serine & Draw the Correct Structure
2:11
Part B: How Many mL of NaOH Solution Have Been Added at This Point (pI)?
5:27
Part C: At What pH is the Average Charge on Serine
10:50
Part D: Draw the Titration Curve for This Situation
14:50
Part E: The 10 mL of NaOH Added to the Solution at the pI is How Many Equivalents?
17:35
Part F: Serine Buffer Solution
20:22
Example 2
23:04
Data
23:05
Part A: Calculate the Minimum Molar Mass of the Protein
25:12
Part B: How Many Tyr Residues in this Protein?
28:34
Example 3
30:08
Question
30:09
Solution
34:30
Example 4
48:46
Question
48:47
Solution
49:50
III. Proteins: Secondary, Tertiary, and Quaternary Structure
Alpha Helix & Beta Conformation

50m 52s

Intro
0:00
Alpha Helix and Beta Conformation
0:28
Protein Structure Overview
0:29
Weak interactions Among the Amino Acid in the Peptide Chain
2:11
Two Principals of Folding Patterns
4:56
Peptide Bond
7:00
Peptide Bond: Resonance
9:46
Peptide Bond: φ Bond & ψ Bond
11:22
Secondary Structure
15:08
α-Helix Folding Pattern
17:28
Illustration 1: α-Helix Folding Pattern
19:22
Illustration 2: α-Helix Folding Pattern
21:39
β-Sheet
25:16
β-Conformation
26:04
Parallel & Anti-parallel
28:44
Parallel β-Conformation Arrangement of the Peptide Chain
30:12
Putting Together a Parallel Peptide Chain
35:16
Anti-Parallel β-Conformation Arrangement
37:42
Tertiary Structure
45:03
Quaternary Structure
45:52
Illustration 3: Myoglobin Tertiary Structure & Hemoglobin Quaternary Structure
47:13
Final Words on Alpha Helix and Beta Conformation
48:34
IV. Proteins: Function
Protein Function I: Ligand Binding & Myoglobin

51m 36s

Intro
0:00
Protein Function I: Ligand Binding & Myoglobin
0:30
Ligand
1:02
Binding Site
2:06
Proteins are Not Static or Fixed
3:36
Multi-Subunit Proteins
5:46
O₂ as a Ligand
7:21
Myoglobin, Protoporphyrin IX, Fe ²⁺, and O₂
12:54
Protoporphyrin Illustration
14:25
Myoglobin With a Heme Group Illustration
17:02
Fe²⁺ has 6 Coordination Sites & Binds O₂
18:10
Heme
19:44
Myoglobin Overview
22:40
Myoglobin and O₂ Interaction
23:34
Keq or Ka & The Measure of Protein's Affinity for Its Ligand
26:46
Defining α: Fraction of Binding Sites Occupied
32:52
Graph: α vs. [L]
37:33
For The Special Case of α = 0.5
39:01
Association Constant & Dissociation Constant
43:54
α & Kd
45:15
Myoglobin's Binding of O₂
48:20
Protein Function II: Hemoglobin

1h 3m 36s

Intro
0:00
Protein Function II: Hemoglobin
0:14
Hemoglobin Overview
0:15
Hemoglobin & Its 4 Subunits
1:22
α and β Interactions
5:18
Two Major Conformations of Hb: T State (Tense) & R State (Relaxed)
8:06
Transition From The T State to R State
12:03
Binding of Hemoglobins & O₂
14:02
Binding Curve
18:32
Hemoglobin in the Lung
27:28
Signoid Curve
30:13
Cooperative Binding
32:25
Hemoglobin is an Allosteric Protein
34:26
Homotropic Allostery
36:18
Describing Cooperative Binding Quantitatively
38:06
Deriving The Hill Equation
41:52
Graphing the Hill Equation
44:43
The Slope and Degree of Cooperation
46:25
The Hill Coefficient
49:48
Hill Coefficient = 1
51:08
Hill Coefficient < 1
55:55
Where the Graph Hits the x-axis
56:11
Graph for Hemoglobin
58:02
Protein Function III: More on Hemoglobin

1h 7m 16s

Intro
0:00
Protein Function III: More on Hemoglobin
0:11
Two Models for Cooperative Binding: MWC & Sequential Model
0:12
MWC Model
1:31
Hemoglobin Subunits
3:32
Sequential Model
8:00
Hemoglobin Transports H⁺ & CO₂
17:23
Binding Sites of H⁺ and CO₂
19:36
CO₂ is Converted to Bicarbonate
23:28
Production of H⁺ & CO₂ in Tissues
27:28
H⁺ & CO₂ Binding are Inversely Related to O₂ Binding
28:31
The H⁺ Bohr Effect: His¹⁴⁶ Residue on the β Subunits
33:31
Heterotropic Allosteric Regulation of O₂ Binding by 2,3-Biphosphoglycerate (2,3 BPG)
39:53
Binding Curve for 2,3 BPG
56:21
V. Enzymes
Enzymes I

41m 38s

Intro
0:00
Enzymes I
0:38
Enzymes Overview
0:39
Cofactor
4:38
Holoenzyme
5:52
Apoenzyme
6:40
Riboflavin, FAD, Pyridoxine, Pyridoxal Phosphate Structures
7:28
Carbonic Anhydrase
8:45
Classification of Enzymes
9:55
Example: EC 1.1.1.1
13:04
Reaction of Oxidoreductases
16:23
Enzymes: Catalysts, Active Site, and Substrate
18:28
Illustration of Enzymes, Substrate, and Active Site
27:22
Catalysts & Activation Energies
29:57
Intermediates
36:00
Enzymes II

44m 2s

Intro
0:00
Enzymes II: Transitions State, Binding Energy, & Induced Fit
0:18
Enzymes 'Fitting' Well With The Transition State
0:20
Example Reaction: Breaking of a Stick
3:40
Another Energy Diagram
8:20
Binding Energy
9:48
Enzymes Specificity
11:03
Key Point: Optimal Interactions Between Substrate & Enzymes
15:15
Induced Fit
16:25
Illustrations: Induced Fit
20:58
Enzymes II: Catalytic Mechanisms
22:17
General Acid/Base Catalysis
23:56
Acid Form & Base Form of Amino Acid: Glu &Asp
25:26
Acid Form & Base Form of Amino Acid: Lys & Arg
26:30
Acid Form & Base Form of Amino Acid: Cys
26:51
Acid Form & Base Form of Amino Acid: His
27:30
Acid Form & Base Form of Amino Acid: Ser
28:16
Acid Form & Base Form of Amino Acid: Tyr
28:30
Example: Phosphohexose Isomerase
29:20
Covalent Catalysis
34:19
Example: Glyceraldehyde 3-Phosphate Dehydrogenase
35:34
Metal Ion Catalysis: Isocitrate Dehydrogenase
38:45
Function of Mn²⁺
42:15
Enzymes III: Kinetics

56m 40s

Intro
0:00
Enzymes III: Kinetics
1:40
Rate of an Enzyme-Catalyzed Reaction & Substrate Concentration
1:41
Graph: Substrate Concentration vs. Reaction Rate
10:43
Rate At Low and High Substrate Concentration
14:26
Michaelis & Menten Kinetics
20:16
More On Rate & Concentration of Substrate
22:46
Steady-State Assumption
26:02
Rate is Determined by How Fast ES Breaks Down to Product
31:36
Total Enzyme Concentration: [Et] = [E] + [ES]
35:35
Rate of ES Formation
36:44
Rate of ES Breakdown
38:40
Measuring Concentration of Enzyme-Substrate Complex
41:19
Measuring Initial & Maximum Velocity
43:43
Michaelis & Menten Equation
46:44
What Happens When V₀ = (1/2) Vmax?
49:12
When [S] << Km
53:32
When [S] >> Km
54:44
Enzymes IV: Lineweaver-Burk Plots

20m 37s

Intro
0:00
Enzymes IV: Lineweaver-Burk Plots
0:45
Deriving The Lineweaver-Burk Equation
0:46
Lineweaver-Burk Plots
3:55
Example 1: Carboxypeptidase A
8:00
More on Km, Vmax, and Enzyme-catalyzed Reaction
15:54
Enzymes V: Enzyme Inhibition

51m 37s

Intro
0:00
Enzymes V: Enzyme Inhibition Overview
0:42
Enzyme Inhibitors Overview
0:43
Classes of Inhibitors
2:32
Competitive Inhibition
3:08
Competitive Inhibition
3:09
Michaelis & Menten Equation in the Presence of a Competitive Inhibitor
7:40
Double-Reciprocal Version of the Michaelis & Menten Equation
14:48
Competitive Inhibition Graph
16:37
Uncompetitive Inhibition
19:23
Uncompetitive Inhibitor
19:24
Michaelis & Menten Equation for Uncompetitive Inhibition
22:10
The Lineweaver-Burk Equation for Uncompetitive Inhibition
26:04
Uncompetitive Inhibition Graph
27:42
Mixed Inhibition
30:30
Mixed Inhibitor
30:31
Double-Reciprocal Version of the Equation
33:34
The Lineweaver-Burk Plots for Mixed Inhibition
35:02
Summary of Reversible Inhibitor Behavior
38:00
Summary of Reversible Inhibitor Behavior
38:01
Note: Non-Competitive Inhibition
42:22
Irreversible Inhibition
45:15
Irreversible Inhibition
45:16
Penicillin & Transpeptidase Enzyme
46:50
Enzymes VI: Regulatory Enzymes

51m 23s

Intro
0:00
Enzymes VI: Regulatory Enzymes
0:45
Regulatory Enzymes Overview
0:46
Example: Glycolysis
2:27
Allosteric Regulatory Enzyme
9:19
Covalent Modification
13:08
Two Other Regulatory Processes
16:28
Allosteric Regulation
20:58
Feedback Inhibition
25:12
Feedback Inhibition Example: L-Threonine → L-Isoleucine
26:03
Covalent Modification
27:26
Covalent Modulators: -PO₃²⁻
29:30
Protein Kinases
31:59
Protein Phosphatases
32:47
Addition/Removal of -PO₃²⁻ and the Effect on Regulatory Enzyme
33:36
Phosphorylation Sites of a Regulatory Enzyme
38:38
Proteolytic Cleavage
41:48
Zymogens: Chymotrypsin & Trypsin
43:58
Enzymes That Use More Than One Regulatory Process: Bacterial Glutamine Synthetase
48:59
Why The Complexity?
50:27
Enzymes VII: Km & Kcat

54m 49s

Intro
0:00
Km
1:48
Recall the Michaelis–Menten Equation
1:49
Km & Enzyme's Affinity
6:18
Rate Forward, Rate Backward, and Equilibrium Constant
11:08
When an Enzyme's Affinity for Its Substrate is High
14:17
More on Km & Enzyme Affinity
17:29
The Measure of Km Under Michaelis–Menten kinetic
23:19
Kcat (First-order Rate Constant or Catalytic Rate Constant)
24:10
Kcat: Definition
24:11
Kcat & The Michaelis–Menten Postulate
25:18
Finding Vmax and [Et}
27:27
Units for Vmax and Kcat
28:26
Kcat: Turnover Number
28:55
Michaelis–Menten Equation
32:12
Km & Kcat
36:37
Second Order Rate Equation
36:38
(Kcat)/(Km): Overview
39:22
High (Kcat)/(Km)
40:20
Low (Kcat)/(Km)
43:16
Practical Big Picture
46:28
Upper Limit to (Kcat)/(Km)
48:56
More On Kcat and Km
49:26
VI. Carbohydrates
Monosaccharides

1h 17m 46s

Intro
0:00
Monosaccharides
1:49
Carbohydrates Overview
1:50
Three Major Classes of Carbohydrates
4:48
Definition of Monosaccharides
5:46
Examples of Monosaccharides: Aldoses
7:06
D-Glyceraldehyde
7:39
D-Erythrose
9:00
D-Ribose
10:10
D-Glucose
11:20
Observation: Aldehyde Group
11:54
Observation: Carbonyl 'C'
12:30
Observation: D & L Naming System
12:54
Examples of Monosaccharides: Ketose
16:54
Dihydroxy Acetone
17:28
D-Erythrulose
18:30
D-Ribulose
19:49
D-Fructose
21:10
D-Glucose Comparison
23:18
More information of Ketoses
24:50
Let's Look Closer at D-Glucoses
25:50
Let's Look At All the D-Hexose Stereoisomers
31:22
D-Allose
32:20
D-Altrose
33:01
D-Glucose
33:39
D-Gulose
35:00
D-Mannose
35:40
D-Idose
36:42
D-Galactose
37:14
D-Talose
37:42
Epimer
40:05
Definition of Epimer
40:06
Example of Epimer: D-Glucose, D-Mannose, and D-Galactose
40:57
Hemiacetal or Hemiketal
44:36
Hemiacetal/Hemiketal Overview
45:00
Ring Formation of the α and β Configurations of D-Glucose
50:52
Ring Formation of the α and β Configurations of Fructose
1:01:39
Haworth Projection
1:07:34
Pyranose & Furanose Overview
1:07:38
Haworth Projection: Pyranoses
1:09:30
Haworth Projection: Furanose
1:14:56
Hexose Derivatives & Reducing Sugars

37m 6s

Intro
0:00
Hexose Derivatives
0:15
Point of Clarification: Forming a Cyclic Sugar From a Linear Sugar
0:16
Let's Recall the α and β Anomers of Glucose
8:42
α-Glucose
10:54
Hexose Derivatives that Play Key Roles in Physiology Progression
17:38
β-Glucose
18:24
β-Glucosamine
18:48
N-Acetyl-β-Glucosamine
20:14
β-Glucose-6-Phosphate
22:22
D-Gluconate
24:10
Glucono-δ-Lactone
26:33
Reducing Sugars
29:50
Reducing Sugars Overview
29:51
Reducing Sugars Example: β-Galactose
32:36
Disaccharides

43m 32s

Intro
0:00
Disaccharides
0:15
Disaccharides Overview
0:19
Examples of Disaccharides & How to Name Them
2:49
Disaccharides Trehalose Overview
15:46
Disaccharides Trehalose: Flip
20:52
Disaccharides Trehalose: Spin
28:36
Example: Draw the Structure
33:12
Polysaccharides

39m 25s

Intro
0:00
Recap Example: Draw the Structure of Gal(α1↔β1)Man
0:38
Polysaccharides
9:46
Polysaccharides Overview
9:50
Homopolysaccharide
13:12
Heteropolysaccharide
13:47
Homopolysaccharide as Fuel Storage
16:23
Starch Has Two Types of Glucose Polymer: Amylose
17:10
Starch Has Two Types of Glucose Polymer: Amylopectin
18:04
Polysaccharides: Reducing End & Non-Reducing End
19:30
Glycogen
20:06
Examples: Structures of Polysaccharides
21:42
Let's Draw an (α1→4) & (α1→6) of Amylopectin by Hand.
28:14
More on Glycogen
31:17
Glycogen, Concentration, & The Concept of Osmolarity
35:16
Polysaccharides, Part 2

44m 15s

Intro
0:00
Polysaccharides
0:17
Example: Cellulose
0:34
Glycoside Bond
7:25
Example Illustrations
12:30
Glycosaminoglycans Part 1
15:55
Glycosaminoglycans Part 2
18:34
Glycosaminoglycans & Sulfate Attachments
22:42
β-D-N-Acetylglucosamine
24:49
β-D-N-AcetylGalactosamine
25:42
β-D-Glucuronate
26:44
β-L-Iduronate
27:54
More on Sulfate Attachments
29:49
Hylarunic Acid
32:00
Hyaluronates
39:32
Other Glycosaminoglycans
40:46
Glycoconjugates

44m 23s

Intro
0:00
Glycoconjugates
0:24
Overview
0:25
Proteoglycan
2:53
Glycoprotein
5:20
Glycolipid
7:25
Proteoglycan vs. Glycoprotein
8:15
Cell Surface Diagram
11:17
Proteoglycan Common Structure
14:24
Example: Chondroitin-4-Sulfate
15:06
Glycoproteins
19:50
The Monomers that Commonly Show Up in The Oligo Portions of Glycoproteins
28:02
N-Acetylneuraminic Acid
31:17
L-Furose
32:37
Example of an N-Linked Oligosaccharide
33:21
Cell Membrane Structure
36:35
Glycolipids & Lipopolysaccharide
37:22
Structure Example
41:28
More Example Problems with Carbohydrates

40m 22s

Intro
0:00
Example 1
1:09
Example 2
2:34
Example 3
5:12
Example 4
16:19
Question
16:20
Solution
17:25
Example 5
24:18
Question
24:19
Structure of 2,3-Di-O-Methylglucose
26:47
Part A
28:11
Part B
33:46
VII. Lipids
Fatty Acids & Triacylglycerols

54m 55s

Intro
0:00
Fatty Acids
0:32
Lipids Overview
0:34
Introduction to Fatty Acid
3:18
Saturated Fatty Acid
6:13
Unsaturated or Polyunsaturated Fatty Acid
7:07
Saturated Fatty Acid Example
7:46
Unsaturated Fatty Acid Example
9:06
Notation Example: Chain Length, Degree of Unsaturation, & Double Bonds Location of Fatty Acid
11:56
Example 1: Draw the Structure
16:18
Example 2: Give the Shorthand for cis,cis-5,8-Hexadecadienoic Acid
20:12
Example 3
23:12
Solubility of Fatty Acids
25:45
Melting Points of Fatty Acids
29:40
Triacylglycerols
34:13
Definition of Triacylglycerols
34:14
Structure of Triacylglycerols
35:08
Example: Triacylglycerols
40:23
Recall Ester Formation
43:57
The Body's Primary Fuel-Reserves
47:22
Two Primary Advantages to Storing Energy as Triacylglycerols Instead of Glycogen: Number 1
49:24
Two Primary Advantages to Storing Energy as Triacylglycerols Instead of Glycogen: Number 2
51:54
Membrane Lipids

38m 51s

Intro
0:00
Membrane Lipids
0:26
Definition of Membrane Lipids
0:27
Five Major Classes of Membrane Lipids
2:38
Glycerophospholipids
5:04
Glycerophospholipids Overview
5:05
The X Group
8:05
Example: Phosphatidyl Ethanolamine
10:51
Example: Phosphatidyl Choline
13:34
Phosphatidyl Serine
15:16
Head Groups
16:50
Ether Linkages Instead of Ester Linkages
20:05
Galactolipids
23:39
Galactolipids Overview
23:40
Monogalactosyldiacylglycerol: MGDG
25:17
Digalactosyldiacylglycerol: DGDG
28:13
Structure Examples 1: Lipid Bilayer
31:35
Structure Examples 2: Cross Section of a Cell
34:56
Structure Examples 3: MGDG & DGDG
36:28
Membrane Lipids, Part 2

38m 20s

Intro
0:00
Sphingolipids
0:11
Sphingolipid Overview
0:12
Sphingosine Structure
1:42
Ceramide
3:56
Subclasses of Sphingolipids Overview
6:00
Subclasses of Sphingolipids: Sphingomyelins
7:53
Sphingomyelins
7:54
Subclasses of Sphingolipids: Glycosphingolipid
12:47
Glycosphingolipid Overview
12:48
Cerebrosides & Globosides Overview
14:33
Example: Cerebrosides
15:43
Example: Globosides
17:14
Subclasses of Sphingolipids: Gangliosides
19:07
Gangliosides
19:08
Medical Application: Tay-Sachs Disease
23:34
Sterols
30:45
Sterols: Basic Structure
30:46
Important Example: Cholesterol
32:01
Structures Example
34:13
The Biologically Active Lipids

48m 36s

Intro
0:00
The Biologically Active Lipids
0:44
Phosphatidyl Inositol Structure
0:45
Phosphatidyl Inositol Reaction
3:24
Image Example
12:49
Eicosanoids
14:12
Arachidonic Acid & Membrane Lipid Containing Arachidonic Acid
18:41
Three Classes of Eicosanoids
20:42
Overall Structures
21:38
Prostagladins
22:56
Thromboxane
27:19
Leukotrienes
30:19
More On The Biologically Active Lipids
33:34
Steroid Hormones
33:35
Fat Soluble Vitamins
38:25
Vitamin D₃
40:40
Vitamin A
43:17
Vitamin E
45:12
Vitamin K
47:17
VIII. Energy & Biological Systems (Bioenergetics)
Thermodynamics, Free Energy & Equilibrium

45m 51s

Intro
0:00
Thermodynamics, Free Energy and Equilibrium
1:03
Reaction: Glucose + Pi → Glucose 6-Phosphate
1:50
Thermodynamics & Spontaneous Processes
3:31
In Going From Reactants → Product, a Reaction Wants to Release Heat
6:30
A Reaction Wants to Become More Disordered
9:10
∆H < 0
10:30
∆H > 0
10:57
∆S > 0
11:23
∆S <0
11:56
∆G = ∆H - T∆S at Constant Pressure
12:15
Gibbs Free Energy
15:00
∆G < 0
16:49
∆G > 0
17:07
Reference Frame For Thermodynamics Measurements
17:57
More On BioChemistry Standard
22:36
Spontaneity
25:36
Keq
31:45
Example: Glucose + Pi → Glucose 6-Phosphate
34:14
Example Problem 1
40:25
Question
40:26
Solution
41:12
More on Thermodynamics & Free Energy

37m 6s

Intro
0:00
More on Thermodynamics & Free Energy
0:16
Calculating ∆G Under Standard Conditions
0:17
Calculating ∆G Under Physiological Conditions
2:05
∆G < 0
5:39
∆G = 0
7:03
Reaction Moving Forward Spontaneously
8:00
∆G & The Maximum Theoretical Amount of Free Energy Available
10:36
Example Problem 1
13:11
Reactions That Have Species in Common
17:48
Example Problem 2: Part 1
20:10
Example Problem 2: Part 2- Enzyme Hexokinase & Coupling
25:08
Example Problem 2: Part 3
30:34
Recap
34:45
ATP & Other High-Energy Compounds

44m 32s

Intro
0:00
ATP & Other High-Energy Compounds
0:10
Endergonic Reaction Coupled With Exergonic Reaction
0:11
Major Theme In Metabolism
6:56
Why the ∆G°' for ATP Hydrolysis is Large & Negative
12:24
∆G°' for ATP Hydrolysis
12:25
Reason 1: Electrostatic Repulsion
14:24
Reason 2: Pi & Resonance Forms
15:33
Reason 3: Concentrations of ADP & Pi
17:32
ATP & Other High-Energy Compounds Cont'd
18:48
More On ∆G°' & Hydrolysis
18:49
Other Compounds That Have Large Negative ∆G°' of Hydrolysis: Phosphoenol Pyruvate (PEP)
25:14
Enzyme Pyruvate Kinase
30:36
Another High Energy Molecule: 1,3 Biphosphoglycerate
36:17
Another High Energy Molecule: Phophocreatine
39:41
Phosphoryl Group Transfers

30m 8s

Intro
0:00
Phosphoryl Group Transfer
0:27
Phosphoryl Group Transfer Overview
0:28
Example: Glutamate → Glutamine Part 1
7:11
Example: Glutamate → Glutamine Part 2
13:29
ATP Not Only Transfers Phosphoryl, But Also Pyrophosphoryl & Adenylyl Groups
17:03
Attack At The γ Phosphorous Transfers a Phosphoryl
19:02
Attack At The β Phosphorous Gives Pyrophosphoryl
22:44
Oxidation-Reduction Reactions

49m 46s

Intro
0:00
Oxidation-Reduction Reactions
1:32
Redox Reactions
1:33
Example 1: Mg + Al³⁺ → Mg²⁺ + Al
3:49
Reduction Potential Definition
10:47
Reduction Potential Example
13:38
Organic Example
22:23
Review: How To Find The Oxidation States For Carbon
24:15
Examples: Oxidation States For Carbon
27:45
Example 1: Oxidation States For Carbon
27:46
Example 2: Oxidation States For Carbon
28:36
Example 3: Oxidation States For Carbon
29:18
Example 4: Oxidation States For Carbon
29:44
Example 5: Oxidation States For Carbon
30:10
Example 6: Oxidation States For Carbon
30:40
Example 7: Oxidation States For Carbon
31:20
Example 8: Oxidation States For Carbon
32:10
Example 9: Oxidation States For Carbon
32:52
Oxidation-Reduction Reactions, cont'd
35:22
More On Reduction Potential
35:28
Lets' Start With ∆G = ∆G°' + RTlnQ
38:29
Example: Oxidation Reduction Reactions
41:42
More On Oxidation-Reduction Reactions

56m 34s

Intro
0:00
More On Oxidation-Reduction Reactions
0:10
Example 1: What If the Concentrations Are Not Standard?
0:11
Alternate Procedure That Uses The 1/2 Reactions Individually
8:57
Universal Electron Carriers in Aqueous Medium: NAD+ & NADH
15:12
The Others Are…
19:22
NAD+ & NADP Coenzymes
20:56
FMN & FAD
22:03
Nicotinamide Adenine Dinucleotide (Phosphate)
23:03
Reduction 1/2 Reactions
36:10
Ratio of NAD+ : NADH
36:52
Ratio of NADPH : NADP+
38:02
Specialized Roles of NAD+ & NADPH
38:48
Oxidoreductase Enzyme Overview
40:26
Examples of Oxidoreductase
43:32
The Flavin Nucleotides
46:46
Example Problems For Bioenergetics

42m 12s

Intro
0:00
Example 1: Calculate the ∆G°' For The Following Reaction
1:04
Example 1: Question
1:05
Example 1: Solution
2:20
Example 2: Calculate the Keq For the Following
4:20
Example 2: Question
4:21
Example 2: Solution
5:54
Example 3: Calculate the ∆G°' For The Hydrolysis of ATP At 25°C
8:52
Example 3: Question
8:53
Example 3: Solution
10:30
Example 3: Alternate Procedure
13:48
Example 4: Problems For Bioenergetics
16:46
Example 4: Questions
16:47
Example 4: Part A Solution
21:19
Example 4: Part B Solution
23:26
Example 4: Part C Solution
26:12
Example 5: Problems For Bioenergetics
29:27
Example 5: Questions
29:35
Example 5: Solution - Part 1
32:16
Example 5: Solution - Part 2
34:39
IX. Glycolysis and Gluconeogenesis
Overview of Glycolysis I

43m 32s

Intro
0:00
Overview of Glycolysis
0:48
Three Primary Paths For Glucose
1:04
Preparatory Phase of Glycolysis
4:40
Payoff Phase of Glycolysis
6:40
Glycolysis Reactions Diagram
7:58
Enzymes of Glycolysis
12:41
Glycolysis Reactions
16:02
Step 1
16:03
Step 2
18:03
Step 3
18:52
Step 4
20:08
Step 5
21:42
Step 6
22:44
Step 7
24:22
Step 8
25:11
Step 9
26:00
Step 10
26:51
Overview of Glycolysis Cont.
27:28
The Overall Reaction for Glycolysis
27:29
Recall The High-Energy Phosphorylated Compounds Discusses In The Bioenergetics Unit
33:10
What Happens To The Pyruvate That Is Formed?
37:58
Glycolysis II

1h 1m 47s

Intro
0:00
Glycolysis Step 1: The Phosphorylation of Glucose
0:27
Glycolysis Step 1: Reaction
0:28
Hexokinase
2:28
Glycolysis Step 1: Mechanism-Simple Nucleophilic Substitution
6:34
Glycolysis Step 2: Conversion of Glucose 6-Phosphate → Fructose 6-Phosphate
11:33
Glycolysis Step 2: Reaction
11:34
Glycolysis Step 2: Mechanism, Part 1
14:40
Glycolysis Step 2: Mechanism, Part 2
18:16
Glycolysis Step 2: Mechanism, Part 3
19:56
Glycolysis Step 2: Mechanism, Part 4 (Ring Closing & Dissociation)
21:54
Glycolysis Step 3: Conversion of Fructose 6-Phosphate to Fructose 1,6-Biphosphate
24:16
Glycolysis Step 3: Reaction
24:17
Glycolysis Step 3: Mechanism
26:40
Glycolysis Step 4: Cleavage of Fructose 1,6-Biphosphate
31:10
Glycolysis Step 4: Reaction
31:11
Glycolysis Step 4: Mechanism, Part 1 (Binding & Ring Opening)
35:26
Glycolysis Step 4: Mechanism, Part 2
37:40
Glycolysis Step 4: Mechanism, Part 3
39:30
Glycolysis Step 4: Mechanism, Part 4
44:00
Glycolysis Step 4: Mechanism, Part 5
46:34
Glycolysis Step 4: Mechanism, Part 6
49:00
Glycolysis Step 4: Mechanism, Part 7
50:12
Hydrolysis of The Imine
52:33
Glycolysis Step 5: Conversion of Dihydroxyaceton Phosphate to Glyceraldehyde 3-Phosphate
55:38
Glycolysis Step 5: Reaction
55:39
Breakdown and Numbering of Sugar
57:40
Glycolysis III

59m 17s

Intro
0:00
Glycolysis Step 5: Conversion of Dihydroxyaceton Phosphate to Glyceraldehyde 3-Phosphate
0:44
Glycolysis Step 5: Mechanism, Part 1
0:45
Glycolysis Step 5: Mechanism, Part 2
3:53
Glycolysis Step 6: Oxidation of Glyceraldehyde 3-Phosphate to 1,3-Biphosphoglycerate
5:14
Glycolysis Step 6: Reaction
5:15
Glycolysis Step 6: Mechanism, Part 1
8:52
Glycolysis Step 6: Mechanism, Part 2
12:58
Glycolysis Step 6: Mechanism, Part 3
14:26
Glycolysis Step 6: Mechanism, Part 4
16:23
Glycolysis Step 7: Phosphoryl Transfer From 1,3-Biphosphoglycerate to ADP to Form ATP
19:08
Glycolysis Step 7: Reaction
19:09
Substrate-Level Phosphorylation
23:18
Glycolysis Step 7: Mechanism (Nucleophilic Substitution)
26:57
Glycolysis Step 8: Conversion of 3-Phosphoglycerate to 2-Phosphoglycerate
28:44
Glycolysis Step 8: Reaction
28:45
Glycolysis Step 8: Mechanism, Part 1
30:08
Glycolysis Step 8: Mechanism, Part 2
32:24
Glycolysis Step 8: Mechanism, Part 3
34:02
Catalytic Cycle
35:42
Glycolysis Step 9: Dehydration of 2-Phosphoglycerate to Phosphoenol Pyruvate
37:20
Glycolysis Step 9: Reaction
37:21
Glycolysis Step 9: Mechanism, Part 1
40:12
Glycolysis Step 9: Mechanism, Part 2
42:01
Glycolysis Step 9: Mechanism, Part 3
43:58
Glycolysis Step 10: Transfer of a Phosphoryl Group From Phosphoenol Pyruvate To ADP To Form ATP
45:16
Glycolysis Step 10: Reaction
45:17
Substrate-Level Phosphorylation
48:32
Energy Coupling Reaction
51:24
Glycolysis Balance Sheet
54:15
Glycolysis Balance Sheet
54:16
What Happens to The 6 Carbons of Glucose?
56:22
What Happens to 2 ADP & 2 Pi?
57:04
What Happens to The 4e⁻ ?
57:15
Glycolysis IV

39m 47s

Intro
0:00
Feeder Pathways
0:42
Feeder Pathways Overview
0:43
Starch, Glycogen
2:25
Lactose
4:38
Galactose
4:58
Manose
5:22
Trehalose
5:45
Sucrose
5:56
Fructose
6:07
Fates of Pyruvate: Aerobic & Anaerobic Conditions
7:39
Aerobic Conditions & Pyruvate
7:40
Anaerobic Fates of Pyruvate
11:18
Fates of Pyruvate: Lactate Acid Fermentation
14:10
Lactate Acid Fermentation
14:11
Fates of Pyruvate: Ethanol Fermentation
19:01
Ethanol Fermentation Reaction
19:02
TPP: Thiamine Pyrophosphate (Functions and Structure)
23:10
Ethanol Fermentation Mechanism, Part 1
27:53
Ethanol Fermentation Mechanism, Part 2
29:06
Ethanol Fermentation Mechanism, Part 3
31:15
Ethanol Fermentation Mechanism, Part 4
32:44
Ethanol Fermentation Mechanism, Part 5
34:33
Ethanol Fermentation Mechanism, Part 6
35:48
Gluconeogenesis I

41m 34s

Intro
0:00
Gluconeogenesis, Part 1
1:02
Gluconeogenesis Overview
1:03
3 Glycolytic Reactions That Are Irreversible Under Physiological Conditions
2:29
Gluconeogenesis Reactions Overview
6:17
Reaction: Pyruvate to Oxaloacetate
11:07
Reaction: Oxaloacetate to Phosphoenolpyruvate (PEP)
13:29
First Pathway That Pyruvate Can Take to Become Phosphoenolpyruvate
15:24
Second Pathway That Pyruvate Can Take to Become Phosphoenolpyruvate
21:00
Transportation of Pyruvate From The Cytosol to The Mitochondria
24:15
Transportation Mechanism, Part 1
26:41
Transportation Mechanism, Part 2
30:43
Transportation Mechanism, Part 3
34:04
Transportation Mechanism, Part 4
38:14
Gluconeogenesis II

34m 18s

Intro
0:00
Oxaloacetate → Phosphoenolpyruvate (PEP)
0:35
Mitochondrial Membrane Does Not Have a Transporter for Oxaloactate
0:36
Reaction: Oxaloacetate to Phosphoenolpyruvate (PEP)
3:36
Mechanism: Oxaloacetate to Phosphoenolpyruvate (PEP)
4:48
Overall Reaction: Pyruvate to Phosphoenolpyruvate
7:01
Recall The Two Pathways That Pyruvate Can Take to Become Phosphoenolpyruvate
10:16
NADH in Gluconeogenesis
12:29
Second Pathway: Lactate → Pyruvate
18:22
Cytosolic PEP Carboxykinase, Mitochondrial PEP Carboxykinase, & Isozymes
18:23
2nd Bypass Reaction
23:04
3rd Bypass Reaction
24:01
Overall Process
25:17
Other Feeder Pathways For Gluconeogenesis
26:35
Carbon Intermediates of The Citric Acid Cycle
26:36
Amino Acids & The Gluconeogenic Pathway
29:45
Glycolysis & Gluconeogenesis Are Reciprocally Regulated
32:00
The Pentose Phosphate Pathway

42m 52s

Intro
0:00
The Pentose Phosphate Pathway Overview
0:17
The Major Fate of Glucose-6-Phosphate
0:18
The Pentose Phosphate Pathway (PPP) Overview
1:00
Oxidative Phase of The Pentose Phosphate Pathway
4:33
Oxidative Phase of The Pentose Phosphate Pathway: Reaction Overview
4:34
Ribose-5-Phosphate: Glutathione & Reductive Biosynthesis
9:02
Glucose-6-Phosphate to 6-Phosphogluconate
12:48
6-Phosphogluconate to Ribulose-5-Phosphate
15:39
Ribulose-5-Phosphate to Ribose-5-Phosphate
17:05
Non-Oxidative Phase of The Pentose Phosphate Pathway
19:55
Non-Oxidative Phase of The Pentose Phosphate Pathway: Overview
19:56
General Transketolase Reaction
29:03
Transaldolase Reaction
35:10
Final Transketolase Reaction
39:10
X. The Citric Acid Cycle (Krebs Cycle)
Citric Acid Cycle I

36m 10s

Intro
0:00
Stages of Cellular Respiration
0:23
Stages of Cellular Respiration
0:24
From Pyruvate to Acetyl-CoA
6:56
From Pyruvate to Acetyl-CoA: Pyruvate Dehydrogenase Complex
6:57
Overall Reaction
8:42
Oxidative Decarboxylation
11:54
Pyruvate Dehydrogenase (PDH) & Enzymes
15:30
Pyruvate Dehydrogenase (PDH) Requires 5 Coenzymes
17:15
Molecule of CoEnzyme A
18:52
Thioesters
20:56
Lipoic Acid
22:31
Lipoate Is Attached To a Lysine Residue On E₂
24:42
Pyruvate Dehydrogenase Complex: Reactions
26:36
E1: Reaction 1 & 2
30:38
E2: Reaction 3
31:58
E3: Reaction 4 & 5
32:44
Substrate Channeling
34:17
Citric Acid Cycle II

49m 20s

Intro
0:00
Citric Acid Cycle Reactions Overview
0:26
Citric Acid Cycle Reactions Overview: Part 1
0:27
Citric Acid Cycle Reactions Overview: Part 2
7:03
Things to Note
10:58
Citric Acid Cycle Reactions & Mechanism
13:57
Reaction 1: Formation of Citrate
13:58
Reaction 1: Mechanism
19:01
Reaction 2: Citrate to Cis Aconistate to Isocitrate
28:50
Reaction 3: Isocitrate to α-Ketoglutarate
32:35
Reaction 3: Two Isocitrate Dehydrogenase Enzymes
36:24
Reaction 3: Mechanism
37:33
Reaction 4: Oxidation of α-Ketoglutarate to Succinyl-CoA
41:38
Reaction 4: Notes
46:34
Citric Acid Cycle III

44m 11s

Intro
0:00
Citric Acid Cycle Reactions & Mechanism
0:21
Reaction 5: Succinyl-CoA to Succinate
0:24
Reaction 5: Reaction Sequence
2:35
Reaction 6: Oxidation of Succinate to Fumarate
8:28
Reaction 7: Fumarate to Malate
10:17
Reaction 8: Oxidation of L-Malate to Oxaloacetate
14:15
More On The Citric Acid Cycle
17:17
Energy from Oxidation
17:18
How Can We Transfer This NADH Into the Mitochondria
27:10
Citric Cycle is Amphibolic - Works In Both Anabolic & Catabolic Pathways
32:06
Biosynthetic Processes
34:29
Anaplerotic Reactions Overview
37:26
Anaplerotic: Reaction 1
41:42
XI. Catabolism of Fatty Acids
Fatty Acid Catabolism I

48m 11s

Intro
0:00
Introduction to Fatty Acid Catabolism
0:21
Introduction to Fatty Acid Catabolism
0:22
Vertebrate Cells Obtain Fatty Acids for Catabolism From 3 Sources
2:16
Diet: Part 1
4:00
Diet: Part 2
5:35
Diet: Part 3
6:20
Diet: Part 4
6:47
Diet: Part 5
10:18
Diet: Part 6
10:54
Diet: Part 7
12:04
Diet: Part 8
12:26
Fats Stored in Adipocytes Overview
13:54
Fats Stored in Adipocytes (Fat Cells): Part 1
16:13
Fats Stored in Adipocytes (Fat Cells): Part 2
17:16
Fats Stored in Adipocytes (Fat Cells): Part 3
19:42
Fats Stored in Adipocytes (Fat Cells): Part 4
20:52
Fats Stored in Adipocytes (Fat Cells): Part 5
22:56
Mobilization of TAGs Stored in Fat Cells
24:35
Fatty Acid Oxidation
28:29
Fatty Acid Oxidation
28:48
3 Reactions of the Carnitine Shuttle
30:42
Carnitine Shuttle & The Mitochondrial Matrix
36:25
CAT I
43:58
Carnitine Shuttle is the Rate-Limiting Steps
46:24
Fatty Acid Catabolism II

45m 58s

Intro
0:00
Fatty Acid Catabolism
0:15
Fatty Acid Oxidation Takes Place in 3 Stages
0:16
β-Oxidation
2:05
β-Oxidation Overview
2:06
Reaction 1
4:20
Reaction 2
7:35
Reaction 3
8:52
Reaction 4
10:16
β-Oxidation Reactions Discussion
11:34
Notes On β-Oxidation
15:14
Double Bond After The First Reaction
15:15
Reaction 1 is Catalyzed by 3 Isozymes of Acyl-CoA Dehydrogenase
16:04
Reaction 2 & The Addition of H₂O
18:38
After Reaction 4
19:24
Production of ATP
20:04
β-Oxidation of Unsaturated Fatty Acid
21:25
β-Oxidation of Unsaturated Fatty Acid
22:36
β-Oxidation of Mono-Unsaturates
24:49
β-Oxidation of Mono-Unsaturates: Reaction 1
24:50
β-Oxidation of Mono-Unsaturates: Reaction 2
28:43
β-Oxidation of Mono-Unsaturates: Reaction 3
30:50
β-Oxidation of Mono-Unsaturates: Reaction 4
31:06
β-Oxidation of Polyunsaturates
32:29
β-Oxidation of Polyunsaturates: Part 1
32:30
β-Oxidation of Polyunsaturates: Part 2
37:08
β-Oxidation of Polyunsaturates: Part 3
40:25
Fatty Acid Catabolism III

33m 18s

Intro
0:00
Fatty Acid Catabolism
0:43
Oxidation of Fatty Acids With an Odd Number of Carbons
0:44
β-oxidation in the Mitochondrion & Two Other Pathways
9:08
ω-oxidation
10:37
α-oxidation
17:22
Ketone Bodies
19:08
Two Fates of Acetyl-CoA Formed by β-Oxidation Overview
19:09
Ketone Bodies: Acetone
20:42
Ketone Bodies: Acetoacetate
20:57
Ketone Bodies: D-β-hydroxybutyrate
21:25
Two Fates of Acetyl-CoA Formed by β-Oxidation: Part 1
22:05
Two Fates of Acetyl-CoA Formed by β-Oxidation: Part 2
26:59
Two Fates of Acetyl-CoA Formed by β-Oxidation: Part 3
30:52
XII. Catabolism of Amino Acids and the Urea Cycle
Overview & The Aminotransferase Reaction

40m 59s

Intro
0:00
Overview of The Aminotransferase Reaction
0:25
Overview of The Aminotransferase Reaction
0:26
The Aminotransferase Reaction: Process 1
3:06
The Aminotransferase Reaction: Process 2
6:46
Alanine From Muscle Tissue
10:54
Bigger Picture of the Aminotransferase Reaction
14:52
Looking Closely at Process 1
19:04
Pyridoxal Phosphate (PLP)
24:32
Pyridoxamine Phosphate
25:29
Pyridoxine (B6)
26:38
The Function of PLP
27:12
Mechanism Examples
28:46
Reverse Reaction: Glutamate to α-Ketoglutarate
35:34
Glutamine & Alanine: The Urea Cycle I

39m 18s

Intro
0:00
Glutamine & Alanine: The Urea Cycle I
0:45
Excess Ammonia, Glutamate, and Glutamine
0:46
Glucose-Alanine Cycle
9:54
Introduction to the Urea Cycle
20:56
The Urea Cycle: Production of the Carbamoyl Phosphate
22:59
The Urea Cycle: Reaction & Mechanism Involving the Carbamoyl Phosphate Synthetase
33:36
Glutamine & Alanine: The Urea Cycle II

36m 21s

Intro
0:00
Glutamine & Alanine: The Urea Cycle II
0:14
The Urea Cycle Overview
0:34
Reaction 1: Ornithine → Citrulline
7:30
Reaction 2: Citrulline → Citrullyl-AMP
11:15
Reaction 2': Citrullyl-AMP → Argininosuccinate
15:25
Reaction 3: Argininosuccinate → Arginine
20:42
Reaction 4: Arginine → Orthinine
24:00
Links Between the Citric Acid Cycle & the Urea Cycle
27:47
Aspartate-argininosuccinate Shunt
32:36
Amino Acid Catabolism

47m 58s

Intro
0:00
Amino Acid Catabolism
0:10
Common Amino Acids and 6 Major Products
0:11
Ketogenic Amino Acid
1:52
Glucogenic Amino Acid
2:51
Amino Acid Catabolism Diagram
4:18
Cofactors That Play a Role in Amino Acid Catabolism
7:00
Biotin
8:42
Tetrahydrofolate
10:44
S-Adenosylmethionine (AdoMet)
12:46
Tetrahydrobiopterin
13:53
S-Adenosylmethionine & Tetrahydrobiopterin Molecules
14:41
Catabolism of Phenylalanine
18:30
Reaction 1: Phenylalanine to Tyrosine
18:31
Reaction 2: Tyrosine to p-Hydroxyphenylpyruvate
21:36
Reaction 3: p-Hydroxyphenylpyruvate to Homogentisate
23:50
Reaction 4: Homogentisate to Maleylacetoacetate
25:42
Reaction 5: Maleylacetoacetate to Fumarylacetoacetate
28:20
Reaction 6: Fumarylacetoacetate to Fumarate & Succinyl-CoA
29:51
Reaction 7: Fate of Fumarate & Succinyl-CoA
31:14
Phenylalanine Hydroxylase
33:33
The Phenylalanine Hydroxylase Reaction
33:34
Mixed-Function Oxidases
40:26
When Phenylalanine Hydoxylase is Defective: Phenylketonuria (PKU)
44:13
XIII. Oxidative Phosphorylation and ATP Synthesis
Oxidative Phosphorylation I

41m 11s

Intro
0:00
Oxidative Phosphorylation
0:54
Oxidative Phosphorylation Overview
0:55
Mitochondrial Electron Transport Chain Diagram
7:15
Enzyme Complex I of the Electron Transport Chain
12:27
Enzyme Complex II of the Electron Transport Chain
14:02
Enzyme Complex III of the Electron Transport Chain
14:34
Enzyme Complex IV of the Electron Transport Chain
15:30
Complexes Diagram
16:25
Complex I
18:25
Complex I Overview
18:26
What is Ubiquinone or Coenzyme Q?
20:02
Coenzyme Q Transformation
22:37
Complex I Diagram
24:47
Fe-S Proteins
26:42
Transfer of H⁺
29:42
Complex II
31:06
Succinate Dehydrogenase
31:07
Complex II Diagram & Process
32:54
Other Substrates Pass Their e⁻ to Q: Glycerol 3-Phosphate
37:31
Other Substrates Pass Their e⁻ to Q: Fatty Acyl-CoA
39:02
Oxidative Phosphorylation II

36m 27s

Intro
0:00
Complex III
0:19
Complex III Overview
0:20
Complex III: Step 1
1:56
Complex III: Step 2
6:14
Complex IV
8:42
Complex IV: Cytochrome Oxidase
8:43
Oxidative Phosphorylation, cont'd
17:18
Oxidative Phosphorylation: Summary
17:19
Equation 1
19:13
How Exergonic is the Reaction?
21:03
Potential Energy Represented by Transported H⁺
27:24
Free Energy Change for the Production of an Electrochemical Gradient Via an Ion Pump
28:48
Free Energy Change in Active Mitochondria
32:02
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Lecture Comments (6)

0 answers

Post by Arrhenius Theory on September 4 at 04:07:03 PM

All enzymatic reactions can be characteried by Michaelis-Menten Kinetics: S ---> P Enzyme.

1 answer

Last reply by: tiffany yang
Thu Dec 5, 2013 1:51 PM

Post by tiffany yang on November 14, 2013

If Ki (dissociation constant for inhibitor) is basically just ligand-protein binding relationship, then Ki will be the ligand concentration when 50% of the enzyme active sites are occupied. Can we apply the same logic for Km?

Can Km be calculated by the concentration of ligand when 50% of enzyme active sites are occupied. From what i remember from lecture, we can't, but i'm not sure why...

0 answers

Post by tiffany yang on November 14, 2013

this is amazing! many many thanks Raffi! everything makes sense now...

1 answer

Last reply by: Professor Hovasapian
Fri Oct 4, 2013 4:04 PM

Post by Tom Beynon on October 4, 2013

Dr Raffi, from down here in Australia, i would like to say thankyou, you are a genuine legend.

Enzymes VII: Km & Kcat

Lecture Slides are screen-captured images of important points in the lecture. Students can download and print out these lecture slide images to do practice problems as well as take notes while watching the lecture.

  • Intro 0:00
  • Km 1:48
    • Recall the Michaelis–Menten Equation
    • Km & Enzyme's Affinity
    • Rate Forward, Rate Backward, and Equilibrium Constant
    • When an Enzyme's Affinity for Its Substrate is High
    • More on Km & Enzyme Affinity
    • The Measure of Km Under Michaelis–Menten kinetic
  • Kcat (First-order Rate Constant or Catalytic Rate Constant) 24:10
    • Kcat: Definition
    • Kcat & The Michaelis–Menten Postulate
    • Finding Vmax and [Et}
    • Units for Vmax and Kcat
    • Kcat: Turnover Number
    • Michaelis–Menten Equation
  • Km & Kcat 36:37
    • Second Order Rate Equation
    • (Kcat)/(Km): Overview
    • High (Kcat)/(Km)
    • Low (Kcat)/(Km)
    • Practical Big Picture
    • Upper Limit to (Kcat)/(Km)
    • More On Kcat and Km

Transcription: Enzymes VII: Km & Kcat

Hello and welcome back to Educator.com, and welcome back to Biochemistry.0000

Today's lesson, we are going to be talking about the Michaelis-Menten constant and this new constant that we are going to introduce called Kcat.0004

This particular lesson is really, really, very, very important.0016

I am not suggesting that the other lessons are not important, but in the biochemistry course, this quantitative study of kinetics is usually, sort of, a daunting thing for the kids.0020

There is just a lot of symbols floating around; there is a lot of mechanisms, a lot of reactions.0035

You have enzyme substrate product, enzyme substrate, enzyme substrate pro...all kinds of things.0040

So, it tends to get really, really confusing.0047

What I wanted to do is I wanted to come back; we have already discussed the Michaelis-Menten kinetics.0051

We have the Michaelis-Menten equation; I wanted to go back and talk about what Km actually means.0056

I want to talk about how it is used in biochemistry, whether that usage is actually justified or not.0062

I just wanted to spend some more time with it- that is it.0070

I just want you to be as comfortable as possible because these things do come up a lot - well, of course, they come up a lot - in the literature, and it is the one area that I think kids are the most...that it alienates them the most easily.0074

It is really not that bad; a lot of it is actually quite intuitive.0089

You just need to, sort of, get past a lot of the symbolism.0093

In any case, let's just jump right on in, and hopefully, we can help you to get a better understanding of what all of these different things mean.0097

Let's see what we can do; OK, let's go ahead and start off with the Michaelis-Menten equation.0104

Again, let's recall the MM - you know what, I am just going to...the Michaelis-Menten equation.0109

It said v0 is equal to Vmax x the substrate concentration over something called Km plus the substrate concentration.0125

Basically, what this equation does is it relates the speed, the velocity, the rate of the reaction, how fast it is going, in either moles per second or molarity per second as a function of substrate concentration, which is really, really, very, very convenient.0138

It is a beautiful, beautiful equation; now, this Vmax, we saw already that if you are given a particular enzyme, you can keep adding substrate, keep adding substrate.0154

You are going to increase the rate, but at some point, all of the enzyme molecules are going to be completely saturated with substrate.0163

All of the binding sites are taken over; now, enzyme can only go so fast.0171

So, no matter how much more substrate you add, you are not going to make it go any faster.0176

From your perspective, what you see - or actually I will draw it on here - is this thing that looks like this.0181

It ends up maxing out at some speed; there is some upper limit to how fast an enzyme can go.0188

You cannot push it any further than it will go if you have a certain amount of enzyme.0193

Now, it is true; you can add more enzyme, but that is experimental conditions.0198

The body does not just add more enzyme; the body needs to keep its enzyme concentration reasonably low because enzymes are not used up in a reaction.0202

It needs to keep them low and constant; it does not just add enzyme whenever it wants to speed it up.0213

It is substrate concentration that changes; Vmax is just an upper limit on how fast the reaction will go.0217

This Km, as it turns out, is remember, it is the concentration at which the speed it 1/2 of the maximum speed.0224

I did not draw it very well here; let me raise this one a little bit, so something like that.0237

That will be Vmax; that is some upper limit that the speed is going to approach.0242

Km is a constant, but it has units of concentration because you remember, it is concentration of substrate that is on the X axis and its rate, its speed that is on the Y axis.0247

So, this Km is the concentration of substrate that I need that will make the enzyme operate at half of its maximum velocity.0262

Half is just a good number- not too low, not too high, right in the middle.0275

That is what Km is, so this is the Michaelis-Menten equation.0280

Now, recall also from the derivation because we did do the derivation, recall that this was derived from the following postulated 2-step process.0284

Michaelis and Menten, they postulated this particular process- a 2-step process.0312

They said that you had enzyme plus substrate.0321

There is an equilibrium that exists between that and the complex enzyme substrate once it is attached, and then, that breaks down into enzyme plus product.0325

Now, there is some k1, which is the rate constant for the forward reaction.0334

There is some k-1 for the breakdown of enzyme substrate back to enzyme and substrate, and then, there is k2, which is the rate constant for the breakdown of the enzyme substrate complex into enzyme and product.0339

They postulated that this was the slow step; this was very important.0353

They postulated that this was the slow step- the rate-determining step.0358

So, when we say slow, we mean rate-determining; I will go ahead and write that down here: the slow step, the rate-determining step.0363

Because again, your reaction is only going to go as fast as your slowest.0372

OK, now, OK, let's see if we can do this.0378

Now, Km is the amount of substrate concentration that will put your enzyme at half velocity.0384

Well, Km is often interpreted as a numerical measure of an enzyme's affinity for its substrate.0392

You will often see Km as interpreted as some numerical measure of an enzyme's affinity for its substrate.0438

I am going to show you why it is interpreted that way, and then, I am going to discuss why you have to be careful with that particular interpretation.0448

For the most part, it is not going to be a problem, as far as the problems that you do in your book or the things that you discuss in class, but it is very, very important not to just automatically assume that some Km value that you are given or that you read off in some reference book for an enzyme is a measure of the affinity of that enzyme for its substrate.0455

I am going to discuss why it is interpreted that way; let's start off there.0478

Here is why.0483

OK, now, in the derivation of the Michaelis-Menten equation, if you want to go back a couple of lessons, the Km was this.0488

It was a combination of the rate constants k1, k2 and k-1.0502

It was k2 + k-1/k1.0506

That was the Km; the actual definition of Km is this.0514

Our interpretation this way makes more sense intuitively because we have a graph that represents it, but this is the actual definition.0518

It is actually a combination of the rate constants of a given process.0527

In this case, you have a 1, 2, 3; you have a 3-step.0531

There is one this way, one this way, one that way; it is a combination of those rate constants.0535

Now, if k2 - let me write this a little bit better - is, in fact, rate-limiting, in other words, if that k2 step is really the slow step as what was postulated, then, k2 is a lot less than k-2.0540

It is just a slow step; the rate constant is very, very low, very, very small compared to k1, this thing.0574

This is small compared to this.0583

Well, because k2 is so much less than k1, we can actually ignore it in the numerator.0592

So, Km becomes Km = k-1/k1.0598

This is what the Km actually becomes under these conditions.0610

OK, now, let's investigate this; what does this mean?0615

When we have the rate constant in the reverse direction, divided by the rate constant in the forward direction, that is the Km under conditions that we have postulated where k2 really is the slow step and can be ignored in the actual definition of Km.0622

Well, here is what it means.0634

Let's go back to our equilibrium.0638

We have enzyme plus substrate in equilibrium with enzyme substrate.0642

This is k1; this is k-1.0648

Now, the forward rate, and you remember, anytime we write a rate law, it is going to be some constant times the concentration of the reactants.0652

In the forward direction, the enzyme and the substrate separately are the reactants.0664

The rate forward is equal to k1 times the enzyme concentration times the substrate concentration.0671

Now, the rate backward, well the rate backward, the reaction is this way.0683

Enzyme substrate complex, that is the reactant.0687

So, the rate backward - excuse me - equals k-1 times the enzyme substrate complex.0691

Well, since, we have an equilibrium, these rates are equal to each other, so let's set them equal to each other.0702

I will do it over here; I will write k1 ES = k-1 ES complex - ES together.0708

OK, now, let me go ahead and rearrange this and actually put k-1 / k1.0722

What I end up with is the following; I will write it on this side.0727

k-1/k1 = enzyme concentration x substrate concentration divided by enzyme substrate complex concentration.0731

So far, so good, OK, notice what this is.0745

This is the equilibrium constant.0753

It happens to be the equilibrium constant for the reverse reaction for...I will write it in the forward direction, though.0764

It is the equilibrium constant for the breakdown of the enzyme substrate complex, ES as reactant, E + S - right - reactants/products.0779

I am sorry, right, no, yes, right.0790

Equilibrium constant is product over reactant; thanks- product over reactant, product over reactant.0792

For this reaction, the breakdown of the enzyme substrate complex, this k-1/k1 is the equilibrium constant.0799

Now, that is what the Km is; the Km actually ends up being an equilibrium constant.0810

Now, when an enzyme's affinity for its substrate is high, that means it is found mostly in the enzyme substrate complex form.0816

It is found in this form; because the enzyme has a high affinity for its substrate, it is going to bind to its substrate, and it wants to be in that form.0840

It does not want to be free enzyme, free substrate.0848

High affinity means high concentration of ES.0852

Let me go back to black here; when an enzyme's affinity for its substrate is high, then, it is found mostly in the ES state.0856

The concentration of ES is very high.0892

OK, well, look what we have; we have Km is equal to, we said, k-1/k1.0899

Well, k-1/k1 is equal to the enzyme concentration times the substrate concentration over the concentration of enzyme substrate.0907

This is just the equilibrium constant, again, for the breakdown of the enzyme substrate complex.0916

Well, if ES is high, that means if the denominator is high, that means the Km is low...is very high, then, Km is going to be low- that is it.0921

Now, this is why we actually interpret or why many biochemists interpret this Km as a measure of affinity for its enzyme.0952

The lower the value of Km, because of this equation that we have right here, that means that the concentration of enzyme substrate complex is very, very high because the enzyme has a high affinity for its substrate.0965

It wants to be bound to it; it does not want to be free enzymes.0981

When this is high, because of how the postulated process works, the Km is going to be very very low.0985

In this particular case, if a mechanism, if the particular enzyme actually follows this Michaelis-Menten postulated process, then, it is true.0993

The Km is a numerical measure of the affinity for an enzyme, for its substrate.1004

The lower the Km, the higher the affinity; again, the Km is just a reflection.1009

It is the amount of substrate you have to add in order to get to half velocity.1015

Well, if all you have to do is add just a little bit of substrate, that means that every little bit of substrate that you add - boom - immediately attaches to the enzyme, and the enzyme is working very, very, very, very fast or doing what it does.1020

You do not have to add a lot of substrate; this is why we interpret Km as the affinity of an enzyme for its substrate.1032

This equation, right here, it is an equilibrium that is existing between the enzyme and the substrate and the enzyme substrate complex.1042

Now, let's go ahead and say a few words about this.1050

Now, many enzymes demonstrate Michaelis-Menten kinetic behavior - OK - but they do not follow the postulated 2-step process that we used to derive the equation.1057

They follow that kinetic behavior; they follow that hyperbolic path.1087

Many enzymes demonstrate Michaelis-Menten kinetic behavior, but they do not follow the postulated 2-step process with step 2 being rate-limiting.1092

Often, Km is a much more complex function of all of the rate constants involved in a given enzyme’s mechanism.1129

You might have 5 different steps.1140

If each one of those steps is involved in some kind of an equilibrium, you have got 10 different rate constants.1144

Km is a lot more complicated than this; often, Km is a much more complex function of the various rate constants for several steps.1148

OK, in these cases, Km is not a simple measure of enzyme affinity for substrate.1180

OK, let's recap what we did; we went through this process, Michaelis-Menten kinetics, using the postulated 2-step process.1220

Again, what we just said, there are many enzymes that actually demonstrate a Michaelis-Menten kinetic behavior.1229

We can find a Km for it; it is not a problem.1236

However, just because it follows Michaelis-Menten kinetic behavior, it does not mean that the enzyme mechanism is that 2-step process that Michaelis and Menten postulated.1240

If it is that simple 2-step process, then yes, we can go ahead and interpret Km as some numerical measure of the affinity for an enzyme for its substrate.1260

The lower the Km, the higher the affinity, but if you are dealing with an enzyme that demonstrates Michaelis-Menten behavior, and if you know something else about that enzymes, let's say you happen to know that the enzyme actually operates with 7 steps or 5 steps or 10 steps or whatever it is, and let's say you are not really sure which one is rate-limiting or let's say even if you do know which one is rate-limiting, the Km at that point, even though you can find it from either the graph or doing a Lineweaver-Burk plot and getting the Km, that is not necessarily going to reflect the enzyme's affinity for its substrate.1268

This idea of interpreting it, this is very, very important.1306

Forgive me if I keep repeating myself; the interpretation of Km as enzyme affinity is based only on this 2-step process.1310

If the 2-step process is satisfied, then yes, you can say that 1 enzyme has a Km here.1317

One enzyme has a Km of 0.5; another enzyme has a Km of 0.2.1323

The enzyme that has a Km of 0.2, I can say, it has a greater affinity for its substrate than the other one, but if it is not the case, just because it demonstrates Michaelis-Menten behavior, is no guarantee that the mechanism is a 2-step process.1328

If that is the case, then, you cannot use Km as a measure of affinity- absolutely not.1343

I am not sure about the extent to which your professor is going to concentrate on this.1350

For all practical purposes, the problems that you do, you are going to go under the assumption that this a simple 2-step process, and we can use Km to decide on affinity.1357

However, understand that when you get into the lab, whether it is undergraduate research that you are doing or whether you just run across this in the future, do not lock it into your head that that is what Km means- it does not.1368

Only for a specific case can you say that this is actually a measure of affinity.1378

OK, now, let's go ahead and talk a little bit more and introduce a new constant called Kcat, which is going to give us a lot of great information, and then, we will connect it to Km; and we will use it as a measure of the efficiency of an enzyme- really, really fantastic.1383

Now, I will do this in blue.1400

Under Michaelis-Menten kinetics, the Km, again, is a measure of how much substrate - this is how we really want to think about it - will bring the rate, will bring the v0 to 1/2 Vmax.1406

It will bring the rate to half maximum velocity.1440

OK, now, I am going to introduce a new constant, something called Kcat, so Kcat.1444

Now, for a reaction with several steps, Kcat is very easy.1455

It is the rate constant for the slowest step, if you happen to know what it is- that simple.1468

For a reaction with several steps, Kcat is the rate constant for the step, which is clearly rate-limiting - OK - for what is obviously the slow step.1472

If you have 2 steps that you are not quite sure, you have got to be careful.1501

If you clearly know which step is actually slowing the reaction down, then, that is going to be your Kcat.1506

OK, for the step, which is clearly rate-limiting...OK, now, from Michaelis-Menten postulate, where we had enzyme + substrate going to enzyme substrate going to enzyme + product, it was k2.1512

That was the postulated slow step; in this particular case, your Kcat = k2.1538

That is easy; now, recall that when we did the derivation for the Michaelis-Menten equation, Vmax, it was defined as k2 times the total enzyme concentration.1545

We know what the total enzyme concentration is; we control that.1564

Now, because Kcat = k2, Vmax = Kcat times...no, I have just substituted the Kcat in for k2.1568

Let me rearrange, and now, I have got Kcat is equal to Vmax over the total enzyme concentration.1588

This is really, really fantastic; this is very, very important equation here.1600

We introduced this thing called the Kcat, which happens to be the same...it is the rate constant for the slowest step in a reaction if I happen to know what that slow step is.1604

In the case of Michaelis-Menten kinetics, I actually have a way of calculating it directly.1614

Vmax, I can get either from the graph or from a Lineweaver-Burk plot, so I have that number.1619

Well, the total enzyme concentration, well, I picked that.1623

Whatever the enzyme concentration is in my particular experiment, I know that.1627

If I take one divided by the other, I have calculated that.1631

I have come up with a way of actually calculating this thing called Kcat; the rate constant for the slowest step in that reaction.1635

This is absolutely fantastic; this is a really, really, really important constant.1642

Well, that is fine; I will go ahead and write it down.1653

Vmax is easily obtainable from a Lineweaver-Burk plot or directly from rate concentration data and ET.1657

Well, it is just the total enzyme concentration, which I control.1682

Now, there is a way of finding Kcat really, really easily.1694

Once you find Vmax, divide by the enzyme total concentration.1699

You have got yourself this Kcat, and we are going to talk a little bit about what this means in, well, right not.1703

Let's do a little bit of unit analysis.1708

Vmax is a velocity; OK, its Vmax units are molarity per second.1712

OK, now, total enzyme concentration is in molarity.1726

This Kcat is equal to molarity per second over molarity.1739

You cancel those, and you get a unit of 1 over second, which you can also do it that way.1747

This is a unit of frequency, the Hertz.1754

This is really, really great; now, Kcat is also called the turnover number, and here is where it is really, really important, why it is a great number, the turnover number.1758

This per second, it means cycles per second.1775

When I get some value of Kcat, that is telling me in 1 second or in 1 minute or in 1 hour, whatever my time unit happens to be, that that is how many cycles the enzyme runs through.1779

One catalytic cycle is one cycle; if I have a Kcat of 600, that means in 1 second, the enzyme goes through 600 cycles.1790

Well, 1 cycle of the enzyme turns over 1 substrate molecule.1800

If the Kcat happens to be 600 that I have calculated for a particular enzyme, that means in 1 second, 600 substrate molecules are being produced, are being turned over- that is it.1805

It is a direct measure of how fast an enzyme is actually working.1816

OK, Kcat is also called the turnover number.1823

Now, OK, it is the number of cycles per second.1828

Again, it is a unit of frequency, of Hertz, and 1 catalytic cycle turns over 1 substrate molecule.1842

Kcat is a direct measure.1866

That is what is great about it; we love direct measures of how many substrate molecules are converted per second- that is it.1873

It is that simple, and again, the time unit does not have to be seconds.1896

It can be minutes; it can be hours.1900

Often, it is going to be seconds or minutes; those are the primary.1903

Kcat is a direct measure of how many substrate molecules are converted per second: 600, 6000, 60000, 5 million- whatever.1905

Now, let's do a little bit more; we said that Vmax, the maximum velocity is equal to Kcat times the total enzyme concentration.1918

The Michaelis-Menten equation, which is v0 = Vmax x substrate concentration over Km + substrate concentration, when I substitute this Vmax, this Kcat ET into Vmax, it becomes v0 = Kcat x total enzyme concentration x substrate concentration over Km + substrate concentration.1934

Now, Kcat, we found a way to take the Michaelis-Menten equation, this new constant that we defined, and we came up with this thing.1977

Now, we have Kcat and Km in the same expression, but it also involves the total enzyme concentration.1987

This is actually really, really great; now, when the substrate concentration is a lot less than Km - OK - when you have low substrate concentration, which is actually going to be the case under a lot of physiological conditions, you are not going to have a lot of substrate molecule floating around all the time.1995

I mean, under experimental conditions that we control, we can control the substrate concentration, but it is not always like that.2017

You have billions and billions of different types of molecules floating around in any given moment in its cell, outside of the cell.2024

The substrate concentration for a given enzyme can actually be a lot lower than the actual concentration that we need to get it to half velocity.2030

So, how does an enzyme actually do what it does?2040

Well, under these conditions, which are pretty standard conditions, watch what happens if S is a lot less than Km.2042

We can actually drop it from this denominator; because it is a lot less than this, it becomes insignificant.2048

Under these conditions, v0 becomes Kcat times the total enzyme concentration times the total substrate concentration over Km, or it becomes this constant, Kcat/Km x enzyme concentration and substrate concentration.2053

Notice, now, our velocity, our rate, is actually has now become a second order reaction.2080

It depends on the concentration of 2 things; it depends on the concentration of enzyme, and it depends on the concentration of substrate.2086

Well, that makes sense because you remember, the reaction is actually this.2095

You have substrate, and you have enzyme.2102

When we write a rate law for this, it should be second order because we have...where both of them are actually controlling the rate.2106

When we write the rate law for this, remember earlier, we wrote it as any rate law is some constant times the concentration of 1 reactant, the concentration of the other reactant.2115

Well, here we go; we have a version of the Michaelis-Menten equation with Kcat, Km that actually shows the dependence on the concentration of both.2125

OK, let me just actually write this here- initial rate.2135

Again, we have the E + S, the ES.2145

This represents the rate when under initial conditions, before actual saturation takes place because that is what we are doing.2151

We are looking at initial rates; that is what we are measuring.2164

This particular version, well, yes, the way we are looking at it actually makes a lot of sense when we are using this Kcat/Km x ET x S because now, it is dependent on both of those things, which is more natural.2168

OK, now, as we approach sat...you know what, actually I am going to write this out.2185

I am sorry; I apologize.2191

I think if I write it out it will make a little bit more sense here.2194

Notice how this rate equation, this v0 equation is, now, second order, which makes sense because again, E + S, you have 2 reactants going to ES.2198

The initial rate before saturation - capital here - depends on both the concentration of the enzyme and the concentration of substrate.2237

Now, as we approach saturation, the concentration of S, now, becomes a lot bigger than Km, so you can drop the Km from the equation.2265

Now, v0 approaches Vmax as expected.2284

Now, Kcat, this Kcat/Km... let me rewrite the equation on this page just so we have it as reference.2304

Actually, you know what, let me do this over because I do not want things to show from previous pages.2316

v0, we said we have Kcat, ET and S/Km + S.2321

Now, as we approach saturation, the concentration of substrate becomes huge compared to the Km.2333

So, we can actually drop the Km from the denominator; when I do that, I end up with just S in the denominator.2340

The Ss cancel my velocity, approaches Kcat ET, which is equal to Vmax.2347

It makes perfect sense; it is in perfect alignment with what is happening under the normal version of the Michaelis-Menten equation.2354

Nothing is changed; now, Kcat/Km - this is the important number - is a number which tells us about the efficiency of the enzyme.2362

Again, we have a way of finding Km from the Lineweaver-Burk plot or from the rate concentration data/graph- whichever we want.2394

We have a way of finding Kcat because it is just a function of the Vmax.2403

We can easily find Kcat and Km; when we take Kcat/Km, that is what is going to give us a nice number.2408

This ratio is what is important.2416

Now, do this one in red; OK, a high Kcat/Km mathematically means a high turnover number.2421

It means a high Kcat and a low Km.2443

In other words, it means high turnover number and a low amount of substrate concentration needed for half velocity, needed for 1/2 of maximum velocity.2453

This is good; this very, very good.2486

This is an efficient enzyme; this is what we want.2490

This is an efficient enzyme.2495

OK, let's talk about what this means; if you have a high Kcat, we said that Kcat is a direct measure of how many substrate molecules are turned over in a given unit of time, let's say per second.2502

If that number is really, really high, that means the enzyme is working very, very fast.2515

Now, a Km, if the Kcat/Km number is high, that means the numerator is high, and the denominator is low.2519

So, Km is low; it means that very, very little, small concentration of substrate is enough to get that enzyme to high velocity, that you can actually get it to the enzyme substrate complex quickly, or in terms of affinity, it has very high affinity for its substrate.2529

That means, for every little substrate molecule that comes around, the enzyme grabs it quickly and turns it over quickly.2550

That is the idea; high affinity is related to Km.2558

High turnover number is related to Kcat; I am sorry.2564

Yes, high affinity, low Km; high turnover number, high Kcat.2568

When those conditions are satisfied, your ratio is very, very high.2573

That is a very efficient enzyme; it turns over quickly, and it does not need a lot of substrate.2577

As the substrate comes in, boom, it is attached to the enzyme, and it is turned over.2585

This is a measure of efficiency; this is a very, very important number: Kcat/Km.2589

OK, now, the other version, if you a have a low Kcat/Km, well, let's just do it one at a time.2595

OK, a low Kcat means low turnover number.2610

In other words, that enzyme does not really turn over a lot of substrate molecules, and a high Km means a high amount of substrate is needed for half maximum velocity.2620

This is not good.2654

This is not good.2659

This is not very efficient.2665

You have to put heavy concentration of substrate in order to get the rate of the reaction to actually get to half velocity.2670

You do not often have a lot of substrate, and if the turnover number, if the Kcat happens to be low anyway on that enzyme, you are going to end up really...it is just not very efficient.2678

It is not turning the enzyme very, very quickly; that is the low Kcat.2690

The Km is high; that means you have to add a lot of substrate just to get it to a point where the reaction actually moves at a reasonable rate.2694

That is not very efficient; when you have a low Kcat and a high Km, your Kcat/Km ratio is going to be small.2700

Here is where Kcat/Km is small because now the denominator is really, really high.2714

The numerator is really, really low, so the ratio is going to be low.2724

This is not efficient; we do not want this.2728

Well, it is not only that we do not want this, under physiological/physio conditions, the concentration of substrate is relatively low.2733

We need an efficient enzyme to catalyze the reactions that are important, and an efficient enzyme is where you have a higher Kcat to Km ratio.2760

High turnover: Kcat; low affinity: Km.2780

Here is the practical big picture.2792

Our practical big picture, if you are given rate concentration data, here is what you do.2803

This is the process- very, very simple.2812

The first thing you want to do is you want to find Km and Vmax either from the graph or from the Lineweaver-Burk plot.2816

I will just say "find Km and Vmax from graph" or better yet, from a Lineweaver-Burk plot.2822

Once you have the Km and the Vmax, find Kcat.2837

I should say "calculate Kcat".2841

Calculate Kcat, which is equal to Vmax divided by the total enzyme concentration.2851

This we get from the graph; this we know.2858

The third step form the ratio Kcat/Km.2862

They sometimes call this thing the specificity constant.2878

You do not necessarily have to know that; what is important is that you are actually able to get Km, Vmax and Kcat, and then, you form Kcat/Km.2882

This Kcat/Km is a measure of how efficient that enzyme is.2897

You want to have a high Kcat, higher efficiency, lower Kcat, low...I am sorry.2902

High Kcat/Km ratio is high efficiency; low Kcat/Km ratio is low efficiency.2908

The Kcat, if that is high, that is a high turnover number.2916

That means lots of substrate molecules per unit time.2920

Km, you want it to be low.2924

That means higher affinity in general under most conditions.2928

That is what is going on there; now, there is an upper limit.2934

There is an upper limit to this Kcat/Km.2940

Again, it just depends on how fast 2 things can actually come together to react, and that upper limit is about 109 per molarity per second.2948

Do not worry about the unit; it is not altogether that important.2962

Now, we will close it off with this, and I will go back to blue.2967

OK, the nice thing about this Kcat/Km is that it can be used to compare efficiencies between different enzymes or the efficiencies of a single enzyme that can have more than 1 substrate, of a single enzyme that takes, that can take - I should be more precise - more than 1 substrate.2973

OK, and here is why; you see, different enzymes...I should say different reactions, the different reactions that the different enzymes catalyze.3050

Different reactions for different enzymes, they have different uncatalyzed rates, the basic rate without catalysis, as well as different mechanisms.3073

Kcat alone or Km alone, they do not provide a reference point for comparison.3110

The only time that you can ever use Km or Kcat alone to compare 2 enzymes or to compare the enzyme with one substrate and the enzyme of another substrate is if the mechanisms is the same and if the uncatalyzed rate of the reaction is the same, if basically all situations are the same.3133

That is what we do in science; we measure things, and we try our best to assign numbers to things that we can measure, but in order to actually have value when we are comparing one measurement of another measurement for different things is if we have a point of reference.3153

Well, because different enzymes have different mechanisms, there is no way to actually...and they have different uncatalyzed reaction rates, well, one reaction rate might speed it up this much, and another reaction rate, which is uncatalyzed might speed it up this much, well they might end up in the same place as far as Kcat or Km is concerned, but the actual difference that it made is going to be different.3166

We cannot really tell when a particular enzyme, how much of a rate enhancement that enzyme has provided for the uncatalyzed reaction.3195

Because we do not have a point of reference, the Km for a given enzyme and the Kcat for a given enzyme alone, I cannot compare the Kms of 2 enzymes or the Kcat of 2 enzymes, but if I take Kcat/Km, that gives me a measure of efficiency; and I can compare that efficiency with the Kcat divided by the Km, the efficiency of another enzyme.3206

Those 2 I cannot compare because now, I brought them to the same reference point.3230

That is why both numbers are important, but together, in my personal opinion, they are more important because you can actually compare different enzymes- really, really great.3235

OK, now, Kcat alone of Km alone does not provide a reference point for comparison, but this Kcat divided by the Km does.3248

It provides a single reference point; now, I can compare 2 enzymes, or I can compare an enzyme with 2 different substrates.3261

It does not matter if the uncatalyzed rates are different.3268

It does not matter if their mechanisms are different; I am concerned with the relative efficiencies of those 2 enzymes.3272

I hope that has helped clear some things up regarding this whole kinetics issue with Km and Vmax and Kcat.3279

Thank you for joining us here at Educator.com; we will see you next time, bye-bye.3286

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