1. Chapter 3. Enzymes 1. Introduction
An enzyme is a protein catalyst that speeds up a reaction without being changed itself.
1) Characteristics of enzyme catalytic reactions:
a) enzyme catalytic reactions are thermodynamically possible. Enzymes accelerate reactions by factors of at least a million, but not change the equilibrium;
b) enzymes are highly specific for their reactants which are so called “substrates”;

2. c) many enzyme catalytic activities are regulated;
d) enzymes possess all physicochemical properties of protein and hence are affected by temperature, pH, organic solvents, or other conditions which denature proteins.

3. 2) Composition of enzyme molecules:
For a conjugated enzyme,
Holoenzyme = apoenzyme + cofactor

4. Cofactor: small organic or inorganic molecule that an enzyme requires for its activity.
Cofactors is divided into two types:
Coenzymes: small organic molecules, usually derived from vitamins. They loosely bind to apoenzymes.
Prosthetic groups: are inorganic ions or organic molecules which are covalently bound to apoenzymes.

5. Some important coenzymes
Coenzymes Function Sources
Nicotinamide adenine transfer H+, e Vit. PP
dinucleotide (NAD+)
dinucleotide phosphate (NADP+)
Flavin adenine dinucleotide (FAD) transfer H Vit. B2
Flavin mononucleotide (FMN) transfer H Vit. B2
Thiamin pyrophosphate (TPP) transfer -CHO Vit. B1
Pyridoxal phosphate transfer -NH2 Vit. B6
Coenzyme A (CoA) transfer acyl Pantothenic
Biocytin transfer CO2 Biotin

6. Some enzymes require both organic and inorganic cofactors, such as cytochrome oxidase which contains heme and Cu.
Some enzymes consist of more than one protein subunit (polypeptide chain) with quaternary structures, such as monomeric, oligomeric and multimeric enzymes.

7. 3) Active site of enzyme: is the region that binds the substrate and converts it into product. The active site must be a 3-d entity which can be bound by substrate(s) via non-covalent weak forces (e.g. salt bridge, H-bond, hydrophobic interaction, Van De Waals force).
The active sites are usually clefts or crevices.

8. Two models for enzyme-substrate binding:
a) Lock-and-key model: the shape of the substrate fits the active site of the enzyme just like a key to its lock.
b) Induced-fit model: interaction between the substrate and the enzyme induces a conformational change in the active site such that the active site is complementary to the substrate.

9. Models for enzyme-substrate binding
(a) Lock-and-key model, (b) Induced-fit model

10. 4) Essential groups in the active site
There are two types of essential groups in the active site of enzyme:
Binding groups: specifically bind to the substrate to form enzyme-substrate complex.
catalytic groups: catalyze conversion of the substrate to product.

12. 5) Substrate specificity of enzymes
The specificity of an enzyme for its substrate depends on the 3-d structure of the active site—only the molecule that binds to the active site to form enzyme-substrate complex can be converted into product.
e.g. trypsin, chymotrypsin, and elastase have different structures of active sites which are specified for their substrates.

13. Enzyme catalytic specificity can be classified into three types:
Absolute specificity: the enzyme can only catalyze one reaction of its specific substrate. e.g.
urease
Urea + H2O NH3 + CO2

14. b) Relative specificity: the enzyme can catalyze a reaction of a group of compounds. e.g.
AA1-AA2…Arg(Lys)-AA(n) -AA (n+1) …
H2O
trypsin
AA1-AA2…Arg(Lys) + AA(n) -AA (n+1) …

15. c) Stereo specificity: the enzyme can only catalyze the reaction of one of the isomers of its specific substrate, such as D or L isomers, cis or trans isomers. e.g.
L-amino acid oxidase
L-amino acid a-keto acid
O NH3 + H2O2

16. Induced fit hypothesis: interaction between substrate and enzyme results in a conformational change at the active site of the enzyme, so that the substrate can bind the enzyme to form a [E-S]complex.

17. Substrate binding sites of three enzymes

18. 6) Classification of enzymes

20. 7) Isoenzymes
Isoenzymes are different forms of an enzyme which catalyze the same reaction but exhibit different physical or kinetic properties.
e.g. several enzymes with different structures catalyze the same reaction showing different pH optimum, substrate affinity, or effect of inhibitors.

21. For example, lactate dehydrogenase (LDH) has five isoenzymes: H4, H3M, H2M2, HM3, and M4. They catalyze the same reaction as following:
LDH
Pyruvate + NADH + H lactate
M subunits predominate in muscle and liver whereas H subunits in the heart.

22. 2. Mechanism of enzymatic catalysis
Thermodynamics: G  E-TS
in which G is the free energy change of a system undergoing a transformation at constant pressure and temperature; E=Eb-Ea (energy change of a system from the start Ea to the end Eb); T is the temperature; S is a measure of the degree of randomness or disorder of a system, called entropy.

23. If G<0, then the reaction can occur spontaneously;
If G=0, then the reaction has reached the equilibrium– no net change can take place;
If G>0, then the reaction can not occur spontaneously, but the reaction can occur when free energy is input.
The G provides no information about the rate of a reaction.

24. 2) Mechanism of enzymatic catalysis: an enzyme raises the reaction rate by stabilizing the transition state of a chemical reaction and decreasing the activation energy of the substrate(s).

26. 3. Enzyme kinetics
Enzyme kinetics is the study of the rates of enzyme-catalyzed reactions.
Enzyme velocity: is the initial rate (V0) of the reaction being catalyzed. The unit of enzyme velocity is mmol/min, or enzyme unit (U).
V0 is usually measured before 10% of the substrate has been converted to product. +

27. V0 changes with time

28. Enzyme activity: refers to the total units of enzyme in the sample.
Enzyme specific activity: refers to the number of enzyme units per mg of protein, or U/mg
Specific activity is a measure of the purity of an enzyme.

29. 1) Factors that affect V0 Enzyme velocity is affected by:
a) Substrate concentration: before the enzyme is saturated by the substrate, increase of substrate concentration [S] will result in increase of the initial velocity. However, after saturation increase of [S] has no effect on V0.
Therefore, the V0-[S] plot is a hyperbolic curve.

30. Effect of [S] on V0

31. b) Enzyme concentration: when the substrate concentration is saturating, increase of the enzyme concentration will lead to increase of V0.
c) Temperature: before the temperature that denature the enzyme, increase of the temperature will raise the enzyme catalytic reaction rate. Optimum temperature: at which the enzyme velocity is the maximum value.

32. d) pH: the pH of the solution affects both the enzyme molecule and the substrate. Too low or too high pH may denature the enzyme.
Optimum pH: the pH at which the enzyme exhibits its maximal activity.

33. Effects of T and pH on V0

34. 2) Michaelis-Menten model
In this model the enzyme-catalytic reactions are proposed as:
k k3
E + S ES E + P k2
“ES” is the enzyme-substrate complex (intermediate).

35. If v=k3 [ES], Vmax=k3 [ET], km=(k2+k3)/k1,
then the following equation may be established:
[S]
v = Vmax
[S] + km
This is the Michaelis-Menten equation, in which Vmax is the maximal rate and km is called Michaelis constant.

36. The meaning of km is expressed as:
When [S]=Km, then v=Vmax/2.
Therefore, Km is equal to the substrate concentration at which the reaction rate is half its maximal value.
The unit of Km is the same as that of [S].

37. 3) The double reciprocal plot
The Vmax and Km can be calculated by the double reciprocal plot: km
V0 Vmax Vmax [S] . = +

39. 4. Enzyme inhibition Inhibition of enzyme activity can be:
Irreversible inhibition—the enzyme activity can not recover even after removal of the inhibitor from the enzyme.
Reversible inhibition—the enzyme will regain its full activity after the inhibitor is removed from the enzyme.
Reversible inhibition includes competitive and noncompetitive inhibition.

40. 1) Irreversible inhibition
Irreversible inhibition refers to the permanent inactivation of the enzyme due to irreversible binding of the inhibitor to the enzyme.
Usually, the irreversible inhibitor covalently or noncovalently binds to the active site of the enzyme with very slow dissociation rate.
e.g. diisopropylphosphofluoridate (DIPF) covalently binds the Ser-OH in the active site of acetylcholinesterase.

41. 2) Reversible competitive inhibition
A competitive inhibitor is structurally similar to the normal substrate for the enzyme. It diminishes the enzyme activity by competing with the substrate to bind to the active site and thus reducing the proportion of enzyme molecules bound to the substrate.
Competitive inhibition can be overcome by increasing the [S].

42. Competitive inhibition+ E S I ES EI
E P

43. Km increases while Vmax remains constant.

44. 3) Reversible noncompetitive inhibition
A noncompetitive inhibitor binds reversibly at a non-active site of the enzyme, causing a conformational change of the enzyme and decrease in catalytic activity.
Unlike competitive inhibition, noncompetitive inhibition can not be overcome by increasing the [S].

45. Non-competitive reactions
E+S ES
E+P
I I
EI+S
EIS
Non-competitive reactions
+ S
－ S +
ESI EI E ES P
Vmax decreases while Km remains constant.

46. 4) Reversible uncompetitive inhibition
A uncompetitive inhibitor binds only to the enzyme-substrate complex and not to the free enzyme. It reduces both the Vmax and Km.

47. Uncompetitive reactions
E+S
E+P ES + I
ESI + E S ES
ESI P

48. Plots of uncompetitive reactions

49. 5. Regulation of enzyme activity
1) Feedback inhibition: refers to the inhibition of an enzyme activity of the early reaction by an end-product of the metabolic pathway.
Sequential feedback inibition—the end-product of one branch of a pathway inhibits the first enzyme after the branchpoint but allows the synthesis of the product of another branch.

50. Feedback inhibition and sequential feedback inhibition

51. 2) Allosteric regulation: the binding of a substrate molecule to one active site affects the binding of substrate molecules to other active sites in the enzyme.
Allosteric enzymes are multisubunit proteins with one or more active sites on each subunit and are regulated by effector molecules (may be substrate or non-substrate molecules). Its characteristic is that the V0-[S] plot gives a sigmoidal curve instead of hyperbolic one.

52. V0-[S] plot of an allosteric enzyme

53. Allosteric activator or inhibitor: increases or decreases the reaction rate catalyzed by the allosteric enzyme, respectively.
3) Other regulations: include chemical modification, proteolytic activation, and regulation of enzyme synthesis and breakdown.