1. How Enzymes Work Online Video Section 6.1, page 192
From McKee and McKee, Biochemistry, 6th Edition, © 2016 by Oxford University Press

2. Enzymes and Activation Energy
Online Video
Enzymes and Activation Energy
Section 6.1, page 193
What is a reaction’s activation energy? How do enzymes influence this quantity?
From McKee and McKee, Biochemistry, 6th Edition, © 2016 by Oxford University Press

3. The Induced Fit Model of Enzyme Catalysis
Online Video
The Induced Fit Model of Enzyme Catalysis
Section 6.1, page 195
What is the induced fit model? How exactly do enzymes change over the course of a reaction?
From McKee and McKee, Biochemistry, 6th Edition, © 2016 by Oxford University Press

4. Cofactors, Coenzymes, and Vitamins
Online Video
Cofactors, Coenzymes, and Vitamins
Section 6.1, page 195
What are cofactors and coenzymes? How do they assist enzymes in catalyzing various reactions?
From McKee and McKee, Biochemistry, 6th Edition, © 2016 by Oxford University Press

5. The Six Types of Enzymes
Online Video
The Six Types of Enzymes
Section 6.2, page 196
The six main types of enzymes found in biological systems.
From McKee and McKee, Biochemistry, 6th Edition, © 2016 by Oxford University Press

6. Enzymatic Inhibition and Lineweaver-Burk Plots
Online Video
Enzymatic Inhibition and Lineweaver-Burk Plots
Section 6.3, page 203
The kinetic basis behind enzymatic inhibition.
From McKee and McKee, Biochemistry, 6th Edition, © 2016 by Oxford University Press

7. An Introduction to Enzymes and Catalysis
Online Video
An Introduction to Enzymes and Catalysis
Section 6.4, page 212
How enzymes catalyze biochemical reactions.
From McKee and McKee, Biochemistry, 6th Edition, © 2016 by Oxford University Press

8. Catalysis Online Video Section 6.4, page 212
From McKee and McKee, Biochemistry, 6th Edition, © 2016 by Oxford University Press

9. Enzyme Regulation Online Video Section 6.5, page 225
From McKee and McKee, Biochemistry, 6th Edition, © 2016 by Oxford University Press

10. Enzyme Regulation: Allosteric Regulation
Online Video
Enzyme Regulation: Allosteric Regulation
Section 6.5, page 227
From McKee and McKee, Biochemistry, 6th Edition, © 2016 by Oxford University Press

11. Enzyme Regulation: Covalent Modification Part 1
Online Video
Enzyme Regulation: Covalent Modification Part 1
Section 6.5, page 226
From McKee and McKee, Biochemistry, 6th Edition, © 2016 by Oxford University Press

12. Enzyme Regulation: Covalent Modification Part 2
Online Video
Enzyme Regulation: Covalent Modification Part 2
Section 6.5, page 226
From McKee and McKee, Biochemistry, 6th Edition, © 2016 by Oxford University Press

13. Chapter 6 Enzymes Overview Section 6.1: Properties of Enzymes
Section 6.2: Classification of Enzymes
Section 6.3: Enzyme Kinetics
Section 6.4: Catalysis
Section 6.5: Enzyme Regulation
Biochemistry in Perspective
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

14. Section 6.1: Properties of Enzymes
Enzymes are undoubtedly the most important molecular machines
To proceed at a viable rate, most reactions require an initial energy input
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

15. Section 6.1: Properties of Enzymes
A chemical reaction occurs when colliding molecules possess a minimum amount of energy called the activation energy (Ea)
More commonly called free energy of activation (DG‡) in biochemistry
Many reactions that are spontaneous (-DG) will proceed at imperceptibly slow rates, because they do not have the energy or correct orientation
The likelihood of a reaction improves with increasing the temperature or using a metal catalyst
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

16. Section 6.1: Properties of Enzymes
Living systems cannot increase temperature without the risk of damaging structures, so they use catalysts (enzymes)
Enzymes can increase reaction rate up to 107 to 1019
Enzymes are also very specific for substrates
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

17. Section 6.1: Properties of Enzymes
Figure 6.1 A Catalyst Reduces the Activation Energy of a Reaction
Catalysts increase reaction rate by lowering activation energy
The free energy of activation (DG‡) is the amount of energy to convert 1 mol of substrate (reactant) from the ground state to the transition state
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

18. Section 6.1: Properties of Enzymes
Each enzyme has a specific active site to bind the substrate
The active site also has amino acid side chains that take an active role in the catalytic process
The active site is used to optimally orient the substrate to achieve the transition state at a lower energy
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

19. Section 6.1: Properties of Enzymes
Some enzymes require certain non-protein components to function: cofactors and coenzymes
Intact functional enzymes with cofactors are holoenzymes
The protein component is the apoenzyme
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

20. Two models of enzyme binding of substrate
Lock and Key
Induced Fit

21. Section 6.2: Classification of Enzymes
International Union of Biochemistry (IUB) instituted a naming convention for enzymes, based upon the type of chemical reaction catalyzed
Six major enzyme categories:
1. Oxidoreductases: Catalyze transfer of electrons
2. Transferases: Transfer functional groups between molecules
3. Hydrolases: Catalyze hydrolysis of a chemical bond
4. Lyases: Catalyze cleavage of C-C, C-O, C-N bonds by other means than by hydrolysis or oxidation
5. Isomerases: Catalyze isomerization of substrate
6. Ligases: Catalyze joining of molecules
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

22. Section 6.2: Classification of Enzymes
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

23. Section 6.3: Enzyme Kinetics
Thermodynamics can predict whether a reaction is spontaneous, but cannot predict rate
The rate or velocity of a reaction is the change of a concentration of reactant or product per unit of time
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

24. Section 6.3: Enzyme Kinetics
Initial velocity (v0) is a velocity at the beginning of a reaction when the concentration of substrate greatly exceeds enzyme concentration
Information about reaction rates is the quantitative study of enzyme catalysis, or enzyme kinetics
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

25. Section 6.3: Enzyme Kinetics
Michaelis-Menten Kinetics
The concept of enzyme substrate complexes:
Introduce the Michaelis constant Km
Km describes the amount of substrate needed for the enzyme to obtain half of its maximum rate of reaction
The lower the value of Km, the greater the affinity of the enzyme for ES complex formation k1
E + S ES E + P
k-1 k2
Km =
k-1 + k2 k1
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

26. Section 6.3: Enzyme Kinetics
Vmax is the maximum velocity a reaction can attain
kcat is the turnover number -- the number of substrate molecule each enzyme site converts to product per unit time.
kcat is Vmax over total enzyme concentration (Et)
Figure 6.4 Initial Reaction Velocity v0 and Substrate Concentration [S] for a Typical Enzyme-Catalyzed Reaction
ν =
Vmax[S]
[S] + Km
Michaelis-Menten Equation
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

27. Section 6.3: Enzyme Kinetics
The specificity constant reflects the relationship between catalytic rate and substrate binding affinity (kcat/Km)
It is a measure of how efficiently an enzyme converts substrates into products
Figure 6.5 A Michaelis-Menten Plot
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

28. Section 6.3: Enzyme Kinetics
Lineweaver-Burk Plots
Using the reciprocal of the Michaelis-Menten equation obtains a more accurate determination of the values
Slope of the line Km/Vmax
1/Vmax is the Y intercept
-1/Km is the X intercept
Figure 6.6 Lineweaver-Burk or Double-Reciprocal Plot
1 𝑉 = 𝐾 𝑚 + 𝑆 𝑉 𝑚𝑎𝑥 𝑆 = 𝐾 𝑚 𝑉 𝑚𝑎𝑥 1 𝑆 𝑉 𝑚𝑎𝑥
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

29. Section 6.3: Enzyme Kinetics
Multisubstrate Reactions
Most reactions involve two or more substrates in two classes:
Sequential—reaction cannot proceed until all substrates are bound to the enzyme active site
Double-Displacement Reactions—first product is released before second substrate binds
Enzyme is altered by first phase of the reaction
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

30. Sequential Mechanism of Multisubstrate Enzymes
Sequential—reaction cannot proceed until all substrates are bound to the enzyme active site

31. Sequential Mechanism of Multisubstrate Enzymes
Double-Displacement Reactions (AKA Ping-Pong)—first product is released before second substrate binds.

32. Section 6.3: Enzyme Kinetics
Enzyme Inhibition
Inhibitors reduce enzyme activity
In living systems inhibitors are important, because they regulate metabolic pathways
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

33. Section 6.3: Enzyme Kinetics
Enzyme inhibition can be reversible or irreversible:
Reversible inhibition can be counteracted by increasing substrate levels or removing the inhibitor
Competitive, noncompetitive, and uncompetitive
Irreversible inhibition occurs when the inhibitor permanently impairs the enzyme (covalent interaction)
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

34. Section 6.3: Enzyme Kinetics
Competitive Inhibitors bind reversibly to the enzyme at the active site, thus competing with substrate binding
Forms enzyme-inhibitor (EI) complex
Increasing substrate concentration overcomes competitive inhibition
Figure 6.8 Michaelis-Menten Plot of Uninhibited Enzyme Activity Versus Competitive Inhibition
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

35. Section 6.3: Enzyme Kinetics
Noncompetitive Inhibitors can bind reversibly to the ES complex at a site other than the active site
Forms EI + S and EIS complex
Changes enzyme conformation
Increased substrate concentration partially reverses inhibition
This is the case for pure noncompetitive inhibition only
Figure 6.10 Michaelis-Menten Plot of Uninhibited Enzyme Activity Versus Noncompetitive Inhibition
Au: Should the last subentry be a main entry, since it is a general statement?
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

36. Section 6.3: Enzyme Kinetics
Uncompetitive Inhibitors: a type of uncompetitive inhibition that involves binding only after substrate is bound
Ineffective at low substrate concentrations
Kinetic Analysis of Enzyme Inhibition: double-reciprocal plots may be used to distinguish competitive, noncompetitive, and uncompetitive inhibition
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

37. Section 6.3: Enzyme Kinetics
Slope of the line Km/Vmax
Competitive inhibition increases Km, (i.e., substrate concentration needed increases) not Vmax (6.10a)
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

38. Section 6.3: Enzyme Kinetics
Slope of the line Km/Vmax
Pure noncompetitive Vmax lowered, Km unchanged
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

39. Section 6.3: Enzyme Kinetics
Slope of the line Km/Vmax
Mixed noncompetitive inhibition—both Vmax and Km change and intersection occur above or below the horizontal axis due to differences in k values
"mixture" of competitive inhibition, in which the inhibitor can only bind the enzyme if the substrate has not already bound, and uncompetitive inhibition, in which the inhibitor can only bind the enzyme if the substrate has already bound
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

40. Section 6.3: Enzyme Kinetics
Slope of the line Km/Vmax
Uncompetitive—Km and Vmax are changed although ratio is the same
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

41. Section 6.3: Enzyme Kinetics
Allosteric Enzymes have a sigmoidal curve rather than a hyperbolic one
Resembles the oxygen-binding curve of hemoglobin
Michaelis-Menten kinetics do not apply to allosteric enzymes
Figure 6.13 The Kinetic Profile of an Allosteric Enzyme
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

42. Section 6.3: Enzyme Kinetics
Enzyme Kinetics, Metabolism, and Macromolecular Crowding
Ultimate goal is understanding enzyme kinetics in living organisms
In vitro work does not always reflect in vivo reality, because cells shows macromolecular crowding, which influences reaction rates and equilibrium constants
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

43. Section 6.4: Catalysis
Scientists use different techniques to understand the catalytic mechanism of enzymes, including:
X-ray crystallography,
chemical inactivation
Computational modeling
reaction mechanism:
is a step-by-step description of a reaction
Electrons flow from a nucleophile to an electrophile
Reactions may take more than step
For each step, a transition state exists between products and reactants
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

44. Section 6.4: Catalysis
Figure 6.15 Energy Diagram for a Two-Step Reaction
One or more intermediates may form during the course of a reaction
Examples of reactive intermediates include free radicals, carbocations, and carbanions
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

45. Section 6.4: Catalysis
Figure 6.15 Energy Diagram for a Two-Step Reaction
In any reaction, only molecules that reach the transition state can convert into product molecules
Stabilizing the transition state lowers energy of activation (Ea) and increases reaction rate
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

46. Effect of Catalyst on Activation Energy
An enzyme catalyst lowers the Activation Energy, thus allowing the reaction to proceed more rapidly, but it only affects reactions that could have happened anyway.

47. Section 6.4: Catalysis Catalytic Mechanisms
Mechanisms of only a few enzymes are known in significant detail
Several factors contribute to enzyme catalysis. The most important are:
Proximity and Strain Effects—the substrate must come in close proximity to the active site
Electrostatic Effects—charge distribution in the largely anhydrous active site may help position the substrate
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

48. Section 6.4: Catalysis
Figure 6.16 Ester Hydrolysis: Hydroxide Ion Catalysis
Acid-Base Catalysis—proton transfer is an important factor in chemical reactions
Hydrolysis of an ester, for example, takes place better if the pH is raised
Hydroxide ion catalysis
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

49. Section 6.4: Catalysis
Figure 6.16 Ester Hydrolysis: General Base Catalysis
More physiological is the use of general bases and acids
Side chains of many amino acids (e.g., histidine, lysine, and aspartate) can be used as general acids or bases
Depends on state of protonation, based on pKa of functional groups
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

50. Section 6.4: Catalysis
Covalent Catalysis—the formation of an unstable covalent bond with a nucleophilic group on the enzyme and an electrophilic group on the substrate
Typical residues used in covalent catalysis are Lys, His, Cys, Asp, Glu, and Ser
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

51. The Roles of Amino Acids in Enzyme Catalysis
Section 6.4: Catalysis
The Roles of Amino Acids in Enzyme Catalysis
The active sites of enzymes are lined with amino acids that create a microenvironment conducive to catalysis
Residues can be catalytic or noncatalytic
In order to participate in catalysis, the amino acid has to be charged or polar
For example, chymotrypsin action in Figure 6.16
Noncatalytic side groups function to orient substrate or stabilize transition state
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

52. The Role of Cofactors in Enzyme Catalysis
Section 6.4: Catalysis
The Role of Cofactors in Enzyme Catalysis
Many proteins require nonprotein cofactors
Metals—important metals in living organisms are alkali metals (Na+, K+, Mg2+, and Ca2+) and transition metals (Zn2+, Fe2+, and Cu2+)
Alkali metals are usually loosely bound and play structural roles
Transition metals usually play a functional role in catalysis as part of a functional group
Metals are good Lewis acids and effective electrophiles
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

53. Section 6.4: Catalysis
Coenzymes—a group of organic molecules that provide enzymes’ chemical versatility
Contain functional groups that amino acid side chains do not
Can be tightly or loosely bound and their structures are often changed by the catalytic process
Most are derived from vitamins
Electron transfer (NAD+)
group transfer (coenzyme A)
high-energy transfer potential (nucleotides)
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

54. Section 6.4: Catalysis
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

55. Effects of Temperature and pH on Enzyme-Catalyzed Reactions
Section 6.4: Catalysis
Effects of Temperature and pH on Enzyme-Catalyzed Reactions
Change in an environmental factor could change enzyme structure and therefore function
Temperature—the higher the temperature, the faster the reaction rate; increased number of collisions
Enzymes are proteins and become denatured at high temperatures
Figure 6.17 The Effect of Temperature on Enzyme Activity
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

56. Section 6.4: Catalysis
Figure 6.18 The Effect of pH on Two Enzymes
pH—hydrogen ion concentration affects enzyme function; therefore, there is a pH optimum
Catalytic activity is related to ionic state of the active site
Changes in ionizable groups could change structure of the enzyme
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

57. Detailed Mechanisms of Enzyme Catalysis
Section 6.4: Catalysis
Detailed Mechanisms of Enzyme Catalysis
Mechanisms of two well-characterized enzymes:
Chymotrypsin—serine protease of 27,000 D
Serine proteases have a triad of amino acids in their active site (e.g., Asp 102, His 57, and Ser 195)
Hydrolyzes peptide bonds adjacent to aromatic amino acids
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

58. Catalysis

59. Section 6.5: Enzyme Regulation
Enzyme regulation is necessary for:
Maintenance of ordered state
Conservation of energy
Responsiveness to environmental changes
Control is accomplished by genetic control, covalent modification (e.g. phosphorylation) , allosteric regulation, and compartmentation
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

60. Section 6.5: Enzyme Regulation
Genetic Control
Genetic control plays an important role in controlling the synthesis of enzymes
Happens at the DNA level and can lead to repression or induction of enzyme synthesis
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

61. Section 6.5: Enzyme Regulation
Covalent Modification
Several covalent modifications in enzyme structure cause changes in function
Types of covalent modification include phosphorylation, methylation, acetylation, and nucleotidylation
Some enzymes produced and stored as proenzymes or zymogens (chymotrypsinogen)
Figure 6.21 The Activation of Chymotrypsinogen
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

62. Section 6.5: Enzyme Regulation
Allosteric Regulation
Enzymes that are regulated by the binding of effectors at allosteric sites
Sigmoidal curve, unlike Michaelis-Menten kinetics
If the effectors are substrates, then it is homotropic; if the ligand is different, then it is heterotropic
Figure 6.22 The Rate of an Enzyme-Catalyzed Reaction as a Function of Substrate Concentration
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

63. Section 6.5: Enzyme Regulation
Figure 6.23a Allosteric Interaction Models
Most allosteric enzymes are multisubunit enzymes
Two theoretical models: concerted and sequential
In the concerted model, all subunits are changed at once from taut (T) to relaxed (R) or vice versa
An activator shifts the equilibrium in favor of the R form; an inhibitor shifts in favor of the T form
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

64. Section 6.5: Enzyme Regulation
Figure 6.23b Allosteric Interaction Models
In the Concerted model, enzyme subunits are connected in such a way that a conformational change in one subunit is necessarily conferred to all other subunits
Concerted model is supported by positive cooperativity where binding of one ligand increases subsequent binding.
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

65. Section 6.5: Enzyme Regulation
Figure 6.23 Allosteric Interaction Models
In the sequential model,
subunits need not exist in the same conformation
molecules of substrate bind via induced-fit protocol
conformational changes are not propagated to all subunits
A more complex model that allows for intermediate formations
Accounts for both positive and negative cooperativity
Neither model perfectly accounts for all enzyme behavior
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press

66. Concerted vs. Sequential Models