1. Chapter 2
Enzyme

2. Contents Properties of enzymes Structural features of enzymes
Mechanism of enzyme-catalyzed reactions
Kinetics of enzyme-catalyzed reactions
Inhibition of enzymes
Regulation of enzymes
Clinical applications of enzymes
Nomenclature

3. Section 1 Properties of Enzymes

4. A + B → C + D § 1.1 General Concepts
spontaneous reaction only if DG is negative.
at equilibrium if DG is zero.
spontaneously impossible if DG is positive.

6. Catalyzed reactions
Reactants need to pass over the energy barrier, G+.
Catalysts reduce the activation energy and assist the reactants to pass over the activation energy.

8. Need for special catalysts
Chemical reactions in living systems are quite different from that in the industrial situations because of
Fragile structures of the living systems
Low kinetic energy of the reactants
Low concentration of the reactants
Toxicity of catalysts
Complexity of the biological systems

9. Enzymes
Enzymes are catalysts that have special characteristics to facilitate the biochemical reactions in the biological systems.
Enzyme-catalyzed reactions take place usually under relatively mild conditions.

10. § 1.2 Characteristics
Enzyme-catalyzed reactions have the following characteristics in comparison with the general catalyzed reactions:
common features: 2 “do” and 2 “don’t”
unique features: 3 “high”

11. Common features
Do not consume themselves: no changes in quantity and quality before and after the reactions.
Do not change the equilibrium points: only enhance the reaction rates.
Apply to the thermodynamically allowable reactions
Reduce the activation energy

12. Unique features
Enzyme-catalyzed reactions have very high catalytic efficiency.
Enzymes have a high degree of specificity for their substrates.
Enzymatic activities are highly regulated in response to the external changes.

13. Activation energy (cal/M)
§ 1.3.a High efficiency
Catalyst
Activation energy (cal/M)
No catalyst
18,000
Normal catalyst
11,700
Hydrogen peroxidase
2,000

14. Accelerated reaction rates
enzyme
Non-enzymatic
rate constant
(kn in s-1)
enzymatic
accelerated reaction rate
Carbonic anhydrase
10-1
106
8 x 106
Chymotrypsin
4 x 10-9
4 x 10-2
Lysozyme
3 x 10-9
5 x 10-1
2 x 108
Triose phosphate isomerase
4 x 10-6
4 x 103
109
Urease
3 x 10-10
3 x 104
1014
Mandelate racemase
5 x 102
1.7 x 1015
Alkaline phosphatase
10-15
102
1017

15. § 1.3.b High specificity
Unlike conventional catalysts, enzymes demonstrate the ability to distinguish different substrates. There are three types of substrate specificities.
Absolute specificity
Relative specificity
Stereospecificity

16. Absolute specificity
Enzymes can recognize only one type of substrate and implement their catalytic functions.

17. Relative specificity
Enzymes catalyze one class of substrates or one kind of chemical bond in the same type.

19. Stereospecificity
The enzyme can act on only one form of isomers of the substrates.
Lactate dehydrogenase can recognize only the L-form but the D-form lactate.

20. § 1.3.c High regulation
Enzyme-catalyzed reactions can be regulated in response to the external stimuli, satisfying the needs of biological processes.
Regulations can be accomplished through varying the enzyme quantity, adjusting the enzymatic activity, or changing the substrate concentration.

21. Section 2 Components of Enzymes

22. § 2.1 Active Center
Almost all the enzymes are proteins having well defined structures.
Some functional groups are close enough in space to form a portion called the active center.
Active centers look like a cleft or a crevice.
Active centers are hydrophobic.

23. Lysozyme
Residues (colored ) in the active site come from different parts of the polypeptide chain .

24. Two essential groups
The active center has two essential groups in general.
Binding group: to associate with the reactants to form an enzyme-substrate complex
Catalytic group: to catalyze the reactions and convert substrates into products

26. Active centers

27. § 2.2 Molecular Components
Simple enzymes: consists of only one peptide chain
Conjugated enzymes:
holoenzyme = apoenzyme cofactor
(protein) (non-protein)
Cofactors: metal ions; small organic molecules

28. Metal ions
Metal-activated enzyme: ions necessary but loosely bound. Often found in metal-activated enzyme.
Metalloenzymes: Ions tightly bound.
Particularly in the active center, transfer electrons, bridge the enzyme and substrates, stabilize enzyme conformation, neutralize the anions.

29. Organic compounds Small size and chemically stable compounds
Transferring electrons, protons and other groups
Vitamin-like or vitamin-containing molecule

30. Coenzymes Prosthetic groups
Loosely bind to apoenzyme. Be able to be separated with dialysis.
Accepting H+ or group and leaving to transfer it to others, or vise versa.
Prosthetic groups
Tightly bind through either covalent or many non-covalent interactions.
Remained bound to the apoenzyme during the course of reaction.

31. Section 3 Mechanism of Enzyme-Catalyzed Reactions

32. To understand the molecular details of the catalyzed reaction.
Proximity and orientation arrangement
Multielement catalysis
Surface effect

33. Lock-and-key model
Both E and S are rigid and fixed, so they must be complementary to each other perfectly in order to have a right match.

34. Induced-fit model
The binding induces conformational changes of both E and S, forcing them to get a perfect match.

35. Hexokinase catalyzing glycolysis
Hexokinase, the first enzyme in the glycolysis pathway, converted glucose to glucose-6-phosphate with consuming one ATP molecule.
Two structural domains are connected by a hinge.
Upon binding of a glucose molecule, domains close, shielding the active site for water.

36. Induced structural changes

37. Section 4 Kinetics of Enzyme- Catalyzed Reactions

38. § 4.1 Reaction rate

39. Initial velocity
The reaction rate is defined as the product formation per unit time.
The slope of product concentration ([P]) against the time in a graphic representation is called initial velocity.
It is of rectangular hyperbolic shape.

40. Reaction velocity curve

41. Intermediate state
Forming an enzyme-substrate complex, a transition state, is a key step in the catalytic reaction.
initial intermediate final

43. Rate constants K1 = rate constant for ES formation
K2 = rate constant for ES dissociation
K3 = rate constant for the product released from the active site

44. § 4.2 Michaelis-Menten Equation
The mathematical expression of the product formation with respect to the experimental parameters
Michaelis-Menten equation describes the relationship between the reaction rate and substrate concentration [S].

45. Assumptions [S] >> [E], changes of [S] is negligible.
K2 is negligible compared with K1.
Steady-state: the rate of E-S complex formation is equal to the rate of its disassociation (backward E + S and forward to E + P)

46. [S] << Km 时，v ∝ [S] [S] >> Km 时，v ≈ Vmax
Describing a hyperbolic curve.
Km is a characteristic constant of E
[S] << Km 时，v ∝ [S]
[S] >> Km 时，v ≈ Vmax

48. Significance of Km
the substrate concentration at which enzyme-catalyzed reaction proceeds at one-half of its maximum velocity
Km is independent of [E]. It is determined by the structure of E, the substrate and environmental conditions (pH, T, ionic strength, …)

50. Km is a characteristic constant of E.
The value of Km quantifies the affinity of the enzyme and the substrate under the condition of K3 << K2. The larger the Km，the smaller the affinity.

51. Km for selected enzymes
Substrate km
Catalase
H2O2 25
Hexokinase
ATP
0.4
D-Glucose
0.05
D-Fructose
1.5
Carbonic anhydrase
HCO3- 9
Chemotrypsin
Glycyltyrosinylglycine
108
N-Benzoyltyrosinamide
2.5
Galactosidase
D-Lactose 4
Threonine dehydratase
L-Threonine 5

52. Significance of Vmax
The reaction velocity of an enzymatic reaction when the binding sites of E are saturated with substrates.
It is proportional to [E].

53. Turnover number k3 = Vmax / [E]
Vmax is the reaction rate when the enzymes are saturated, and is independent of the enzyme concentration.
The number of the products converted in a unit time by one enzyme molecule which is saturated.

54. Lineweaver-Burk plot To determine Km and Vmax
To identify the reversible repression

55. Double-reciprocal plot

56. § 4.3 Factors affecting enzyme-catalyzed reaction
Substrate concentration
Enzyme concentration
Temperature pH
Inhibitors
Activators

57. § 4.3.a Effect of substrate
Has been described already.

58. § 4.3.b Effect of enzyme
[E] affects the rate of enzyme-catalyzed reactions
[S] is held constant.
When [S] >> [E], V ≈ [E]

59. § 4.3.c Effect of temperature
Optimal temperature (To) is the characteristic T at which an enzyme has the maximal catalytic power.
35 ~ 40C for warm blood species.
Reaction rates increase by 2 folds for every 10C rise.
Higher T will denature the enzyme.

61. § 4.3.d Effect of pH
Optimal pH is the characteristic pH at which the enzyme has the maximal catalytic power.
pH7.0 is suitable for most enzymes.
Particular examples:
pH (pepsin) = 1.8
pH (trypsin) = 7.8

63. Section 5 Inhibition of Enzyme

64. § 5.1 Inhibitors
Inhibitors are certain molecules that can decrease the catalytic rate of an enzyme-catalyzed reaction.
Inhibitors can be normal body metabolites and foreign substances (drugs and toxins).

65. Inhibition processes
The inhibition process can be either irreversible or reversible.
The inhibition can be competitive, non-competitive, or un-competitive.

66. § 5.2 Irreversible inhibition
Inhibitors are covalently bound to the essential groups of enzymes.
Inhibitors cannot be removed with simple dialysis or super-filtration.
Binding can cause a partial loss or complete loss of the enzymatic activity.

67. Pesticide poisoning
Acetylcholine accumulation will cause excitement of the parasympathetic system: omitting, sweating, muscle trembling, pupil contraction

70. Heavy metal poisoning
Heavy metal containing chemicals bind to the –SH groups to inactivate the enzymes.

72. § 5.3 Reversible inhibition
Inhibitors are bound to enzymes non-covalently.
The reversible inhibition is characterized by an equilibrium between free enzymes and inhibitor-bound enzymes.

73. § 5.3.a Competitive inhibition

74. Competitive inhibitors share the structural similarities with that of substrates.
Competitive inhibitors compete for the active sites with the normal substrates.
Inhibition depends on the affinity of enzymes and the ratio of [E] to [S].

76. Lineweaver-Burk plot

77. Inhibition features
As [S] increases, the effect of inhibitors is reduced, leading to no change in Vmax.
Due to the competition for the binding sites, Km rises, equivalent to the reduction of the affinity.

78. Example-1: competitive inhibitor
FH4 (tetrahydrofolate) is a coenzyme in the nucleic acid synthesis, and FH2 (dihydrofolate) is the precursor of FH4.
Bacteria cannot absorb folic acid directly from environment.
Bacteria use p-amino-benzoic acid (PABA), Glu and dihydropterin to synthesize FH2.
Sulfanilamide derivatives share the structural similarity with PABA, blocking the FH2 formation as a competitive inhibitor.

80. Example-2: competitive inhibitor

82. § 5.3.b Non-competitive inhibition

83. Inhibitors bind to other sites rather than the active sites on the free enzymes or the E-S complexes.
The E-I complex formation does not affect the binding of substrates.
The E-I-S complexes do not proceed to form products.
Reducing the [E-S]
Vmax↓; unchanged Km.

85. § 5.3.c Uncompetitive inhibition

86. Uncompetitive inhibitors bind only to the enzyme-substrate complexes.
The E-I-S complexes do not proceed to form products.
The E-I-S complexes do not backward to the substrates and enzymes.
This inhibition has the effects on reducing both Vmax and Km.
Commonly in the multiple substrate reactions.

88. Summary of inhibition type binding target Km Vmax Competitive E only =
Noncompetitive
E or ES 
Uncompetitive
ES only

89. Activator
Activators are the compounds which bind to an enzyme or an enzyme-substrate complex to enhance the enzymatic activity without being modified by the enzymes.

91. Activators
Metal ions
essential activators: no enzymatic activity without it
Mg2+ of hexokinase
non-essential activators: enhancing the catalytic power.

92. Enzymatic activity
Enzymatic activity is a measure of the capability of an enzyme of catalyzing a chemical reaction.
It directly affects the reaction rate.
International unit (IU): the amount of enzyme required to convert 1 µmol of substrate to product per minute under a designated condition.

93. Determination of the enzymatic activity requires proper treatment of enzymes, excess amount of substrate, optimal T and pH, …
One katal is the amount of enzyme that converts 1 mol of substrate per second.
IU = 16.67×10-9 kat

95. § 2.2 Molecular Components
In addition to enzymes, other chemical species often participate in the catalysis.
Cofactor: chemical species required by inactive apoenzymes (protein only) to convert themselves to active holoenzymes.

96. Cofactors

97. Essential ions
Activator ions: loosely and reversibly bound, often participate in the binding of substrates.
Metal ions of metalloenzymes: tightly bound, and frequently participate directly in catalytic reactions.

98. Function of metal ions Transfer electron Linkage of S and E；
Keep conformation of E-S complex
Neutralize anion

99. Coenzymes
Act as group-transfer reagents to supply active sites with reactive groups not present on the side chains of amino acids
Cosubstrates:
Prosthetic groups:

100. Cosubstrates The substrates in nature.
Their structures are altered for subsequent reactions.
Shuttle mobile metabolic groups among different enzyme-catalyzed reactions.

101. Prosthetic groups
Supply the active sites with reactive groups not present on the side chains of AA residues.
Can be either covalently attached to its apoenzymes or through many non-covalent interactions.
Remained bound to the enzyme during the course of the reaction.

102. Coenzymes
Metabolite coenzymes: they are synthesized from the common metabolites.
several NTP, ATP (most abundant), UDP-glucose
Vitamin-derived coenzymes: they are derivatives of vitamins, and can only be obtained from nutrients.
NAD and NADP+, FAD and FMN, lipid vitamins, …

103. § 2.3 Ribozyme
Until recently, all the enzymes are known to be proteins.
Ribonucleic acids also demonstrate the catalytic ability.
Ribozymes have the ability to self-cleave.
They are highly conservative, an indication of the biological evolution and the primary enzyme.

104. Family of serine protease

105. § 5.3.a Competitive inhibition

106. § 5.3.b Non-competitive inhibition

107. § 5.3.c Uncompetitive inhibition

108. Section 6 Regulation of Enzyme

109. Many biological processes take place at a specific time; at a specific location and at a specific speed.
The catalytic capacity is the product of the enzyme concentration and their intrinsic catalytic efficiency.
The key step of this process is to regulate either the enzymatic activity or the enzyme quantity.

110. Reasons for regulation
Maintenance of an ordered state in a timely fashion and without wasting resources
Conservation of energy to consume just enough nutrients
Rapid adjustment in response to environmental changes

111. Controlling an enzyme that catalyzes the rate-limiting reaction will regulate the entire metabolic pathway, making the biosystem control more efficient.
Rate limiting reaction is the reaction whose rate set by an enzyme will dictate the whole pathway, namely, the slowest one or the “bottleneck” step.

112. §6.1 Regulation of E Activity
Zymogen activation
Allosteric regulation
Covalent modification

113. §6.1.a Zymogen activation
Certain proteins are synthesized and secreted as an inactive precursor of an enzyme, called zymogen.
Selective proteolysis of these precursors leads to conformational changes, and activates these enzymes.
It is the conformational changes that either form an active site of the enzyme or expose the active site to the substrates.

114. Wide varieties Hormones: proinsulin Digestive proteins: trypsinogen, …
Funtional proteins: factors of blood clotting and clot dissolution
Connective tissue proteins: procollagen

115. Activation of chymotrypsin

116. Features of zymogen activation
A cascade reaction in general
To protect the zymogens from being digested
To exert function in appropriate time and location
Store and transport enzymes

117. §6.1.b Allosteric regulation
Allosteric enzymes are those whose activity can be adjusted by reversible, non-covalent binding of a specific modulator to the regulatory sites, specific sites on the surface of enzymes.
Allosteric enzymes are normally composed of multiple subunits which can be either identical or different.

118. The multiple subunits are catalytic subunits regulatory subunits
Kinetic plot of v versus [S] is sigmoidal shape.
Demonstrating either positive or negative cooperative effect.

119. Properties of allosteric enzymes
There are two conformational forms, T and R, which are in equilibrium.
Modulators and substrates can bind to the R form only; the inhibitors can bind to the T form.

120. Allosteric curve

121. Activation of protein kinase
C: catalytic portions
R: regulatory portions

122. §6.1.c Covalent modification
A variety of chemical groups on enzymes could be modified in a reversible and covalent manner.
Such modification can lead to the changes of the enzymatic activity.

123. Common modifications phosphorylation - dephosphorylation
adenylation - deadenylation
methylation - demethylation
uridylation - deuridylation
ribosylation - deribosylation
acetylation - deacetylation

124. Phosphorylation

125. Features of covalent modification
Two active forms (high and low)
Covalent modification
Energy needed
Amplification cascade
Some enzymes can be controlled by allosteric and covalent modification.

126. §6.2 Regulation of E Quantity
Constitutive enzymes (house-keeping): enzymes whose concentration essentially remains constant over time
Adaptive enzymes: enzymes whose quantity fluctuate as body needs and well-regulated.
Regulation of enzyme quantity is accomplished through the control of the genes expression.

127. Controlling the synthesis
Inducer: substrates or structurally related compounds that can initiate the enzyme synthesis
Repressor: compounds that can curtail the synthesis of enzymes in an anabolic pathway in response to the excess of an metabolite
Both are cis elements, trans-acting regulatory proteins, and specific DNA sequences located upstream of genes

128. Controlling the degradation
Enzymes are immortal, and have a wide range of lifetime. LDH4 5-6 days, amylase 3-5 hours.
They degrade once not needed through proteolytic degradation.
The degradation speed can be influenced by the presence of ligands such as substrates, coenzymes, and metal ions, nutrients and hormones.

129. Degradation pathway Lysosomic pathway: Non-lysosomic pathway:
Under the acidic condition in lysosomes
No ATP required
Indiscriminative digestion
Digesting the invading or long lifetime proteins
Non-lysosomic pathway:
Digest the proteins of short lifetime
Labeling by ubiquitin followed by hydrolysis
ATP needed

130. Enzymes/pathways in cellular organelles
Enzyme/metabolic pathway
Cytoplasm
Aminotransferases, peptidases, glycolysis, hexose monophosphate shunt, fatty acids synthesis, purine and pyrimidine catabolism
Mitochondria
Fatty acid oxidation, amino acid oxidation, Krebs cycle, urea synthesis, electron transport chain and oxidative phosphorylation
Nucleus
Biosynthesis of DNA and RNA
Endoplasmic reticulum
Protein biosynthesis, triacylglycerol and phospholipids synthesis, steroid synthesis and reduction, cytochrome P450, esterase
Lysosomes
Lysozyme, phosphatases, phospholipases, proteases, lipases, nucleases
Golgi apparatus
Glucose 6-phosphatase, 5’-nucleotidase, glucosyl- and galactosyl-transferase
Peroxisomes
Calatase, urate oxidase, D-amino acid oxidase, long chain fatty acid oxidase

131. Section 7 Clinical Applications

132. §7.1 Fundamental Concepts
Plasma specific or plasma functional enzymes: Normally present in the plasma and have specific functions.
High activities in plasma than in the tissues. Synthesized in liver and enter the circulation.
Impairment in liver function or genetic disorder leads to a fall in the activities.

133. Non-plasma specific or plasma non-functional enzymes: either totally absent or at a low concentration in plasma
In the normal turnover of cells, intracellular enzymes are released into blood stream.
An organ damaged by diseases may elevate those enzymes

134. §7.2 Isoenzyme
A group of enzymes that catalyze the same reaction but differ from each other in their structure, substrate affinity, Vmax, and regulatory properties.
Due to gene differentiation: the different gene products or different peptides of the same gene
Present in different tissues of the same system, or subcellular components of the same cell

135. Reasons for isoenzyme
Synthesized from different genes (malate dehydrogenase in cytosol versus in mitochondria)
Oligomeric forms of more than one type of subunits (lactate dehydrogenase)
Different carbohydrate content (alkaline phosphatase)

136. Lactate dehydrogenase (LDH)
5 isoenzymes, LDH1 – LDH5
Tetramer
M subunits (M for muscle), basic
H subunits (H for heart), acidic
Different catalytic activities
Used as the marker for disease diagnosis

138. LDH1 (H4) in heart muscle converts lactate to pyruvate, and then to acetyl CoA.
LDH5 (M4) in skeletal muscle converts pyruvate to lactate.

139. Creatine phosphokinase
3 isoenzymes, BB, BM, MM
Dimeric form: M (muscle) or B (brain)
CPK2 is undetectable (<2%) in serum for healthy individuals, and elevated to 20% in the first 6-18 hrs after myocardial infarction.
Used as a earliest reliable indicator of myocardial infarction.

140. §7.3 Diagnostic Applications
Usefulness:
Enzyme assays provide important information concerning the presence and severity of diseases
Provide a means of monitoring the patient’s response
approaches:
Measuring the enzymatic activities directly
Used as agents to monitor the presence of substrates

141. Enzymatic activity changes

142. Electrophoresis of LDH

143. Enzymes for disease diagnosis
Serum enzymes
(elevated)
Diseases
Amylase
Acute pancreatitis
Serum glutamate pyruvate transaminase (SGPT)
Liver diseases (hepatitis)
Serum glutamate oxaloacetate transaminase (SGOT)
Heart attack (myocardial infarction)
Alkaline phosphatase
Rickets, obstructive jaundice
Acid phosphatase
Cancer of prostate gland
Lactate dehydrogenase (LDH)
Heart attack, liver diseases
γ-glutamyl transpeptidase (GGT)
Alcoholism
5’-nucleotidase
Hepatitis
Aldolase
Muscular dystrophy

144. §7.4 Therapeutic Applications
Successful therapeutic uses
Steptokinase: treating myocardial infarction; preventing the heart damage once administrated immediately after heart attack
Asparaginase: tumor regression
Several limits
Can be rapidly inactivated or digested
May provoke allergic effects

145. Section 8 Nomenclature

146. §8.1 Conventional Nomenclature
Adding the suffix –ase to the name of the substrates (urease)
Adding the suffix –ase to a descriptive term for the reactions they catalyze (glutemate dehydrogenase)
For historic names (trypsin, amylase)
Being named after their genes (Rec A –recA, HSP70)

147. §8.2 Systematic Nomenclature
The International Union of Biochemistry and Molecular Biology (IUBMB) maintains the classification scheme.
Categorize in to 6 classes according to the general class of organic reactions catalyzed
Assigned a unique number, a systematic name, a shorter common name to each enzyme

148. §8.2.a Oxidoreductases
Catalyzing a variety of oxidation-reduction reactions
AH2 + B → A + BH2
Alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, E.C )
Cytochrome oxidase
L- and D-amino acid oxidase

149. §8.2.b Transferases
Catalyzing transfer of a groups between donors and acceptors
A-X + B → A + B-X
Hexokinase (ATP:D-hexose 6-phosphotransferase, E.C )
Transaminase
Transmethylases

150. §8.2.c Hydrolases Catalyzing cleavage of bonds by addition of water
A-B + H2O → AH + BOH
Lipase (triacylglycerol acyl hydrolase, E.C )
Choline esterase
Acid and alkaline phosphatases
Urease

151. §8.2.d Lysases
Catalyzing lysis of a substrate and generating a double bond (nonhydrolytic, and non-oxidative reactions)
A-B + X-Y → AX + BY
Aldolase (ketose 1-phosphate aldehyde lysase, E.C )
Fumarase
Histidase

152. §8.2.e Isomerases
Catalyzing recemization of optical or geometric isomers
A → A’
Triose phosphate isomerase (D-glyceraaldehyde 3-phosphate ketoisomerase, E.C )
Retinol isomerase
Phosphohexose isomerase

153. §8.2.f Ligases
Catalyzing synthetic reactions at the expense of a high energy bond of ATP
A + B → A-B
Glutamine synthetase (L-glutamate ammonia ligase, E.C )
Acetyl CoA carboxylase
Auccinate thiokinase

154. Blood clot formation and tissue repair are brought “on-line” only in response to pressing physiological or pathophysiological needs.