1. Chapter 6&7. The Behavior of Proteins.

2. The Behavior of Proteins: Enzymes
Chapter 6
The Behavior of Proteins: Enzymes
- The enzymes: the most remarkable and highly specialized proteins
 They have a high degree of specificity for their substrates
 They accelerate chemical reactions tremendously (by the factor of up to 1020)
 They function in aqueous solutions under mild conditions of Temp. and pH
- The importance of enzyme
 In some diseases, there may be a deficiency of one or more enzymes
 For other diseases, excessive activity of an enzyme may be the cause
* Measurements of the enzyme activity are important in diagnosing diseases
* Many drugs exert their biological effects through interactions with enzymes
* Enzymes are important practical tools, not only in medicine but in chemical industry, food processing, and agriculture

3. Kinetics vs Thermodynamics
Chapter 6
Kinetics vs Thermodynamics
- The reaction rate depends on the free energy of activation (∆Gº)

4. Kinetics vs Thermodynamics
Chapter 6
Kinetics vs Thermodynamics
- Example of the effect of catalysts on activation energy: 2H2O2  2H2O + O2
- Will a reaction go faster if you raise the temperature?
 Increase in the rate with T occurs only to a limited extent
 Heat denaturation of enzyme slows down the reaction

5. Kinetics vs Thermodynamics
Chapter 6
Kinetics vs Thermodynamics
Reaction Rates Have Precise Thermodynamic Definitions
- The rate of the reaction is determined by the concentration of the reactant and by a rate constant, usually denoted by k
For unimolar reaction S  P, the rate of the reaction (V) representing the amount of S that reacts per second is expressed by a rate equation:
V = k[S] : The first-order reaction
, k = a first-order rate constant and has units of reciprocal time, such as s-1
 If k = 0.03s-1, this means that 3% of the substrates will be converted to P in 1 s
- If a reaction rate depends on the concentration of two different compounds, k is a second-order rate constant, with units of M-1s-1
V = k[S1][S2]

6. Enzyme-Substrate Binding
Chapter 6
Enzyme-Substrate Binding
- The distinguishing feature of an enzyme-catalyzed reaction is that it takes place within the confines of a pocket on the enzyme called the active site
- The molecule that is bound in the active site and acted upon by the enzyme is called the substrate
- The surface of the active site is lined with amino acid residues
with substituent groups that bind the substrate
and catalyze its chemical transformation

7. Enzyme-Substrate Binding
Chapter 6
Enzyme-Substrate Binding
- Emil Fischer proposed in 1894 that enzymes were structurally complementary to their substrates, so that they fit together like a lock and key
 The “lock and key” hypothesis can be misleading when applied to enzymatic catalysis
 An enzyme completely complementary to its substrate would be a very poor enzyme

8. Enzyme-Substrate Binding
Chapter 6
Enzyme-Substrate Binding
- If the binding of E and S to form ES were a perfect fit, the ES would be at such a low energy that the difference between ES and EX would be very large
 In general, enzymes increase the rate by lowering the energy of the transition state (EX), while raising the energy of the ES complex

9. Examples of Enzyme-Catalyzed Reactions
Chapter 6
Examples of Enzyme-Catalyzed Reactions
- Chymotrypsin is an enzyme that catalyzes the hydrolysis of peptide bonds for residues containing aromatic side chains. It also catalyzes the hydrolysis of ester bonds
 The curve is hyperbolic (for most enzymes)

10. Examples of Enzyme-Catalyzed Reactions
Chapter 6
Examples of Enzyme-Catalyzed Reactions
- Aspartate transcarbamoylase is used to make carbamoyl aspartate
 The curve is sigmoidal (for most allosteric proteins )
Carbamoyl phosphate + Aspartate
 Carbamoyl aspartate + HPO4-2

11. The Michaelis-Menten Approach to Enzyme Kinetics
Chapter 6
The Michaelis-Menten Approach to Enzyme Kinetics
Substrate Concentration Affects the Rate of Enzyme-Catalyzed Reactions
- The substrate concentration, [S], is the key factor for the enzyme-catalyzed reaction
V0: the initial rate, Vmax: maximum velocity

12. The Michaelis-Menten Approach to Enzyme Kinetics
Chapter 6
The Michaelis-Menten Approach to Enzyme Kinetics
- Theory by Leonor Michaelis and Maud Menten in 1913
 The enzyme first combine reversibly with its substrate to form a complex
E + S ↔ ES (k1, k-1) : Relatively fast reversible step
 The ES complex then breaks down
ES ↔ E + P (k2, k-2) : The slower reaction
 The overall rate is proportional to the concentration of ES
 The maximum initial rate of the reaction is observed
when all the enzyme is present as the ES complex
and [E] is small

13. The Michaelis-Menten Approach to Enzyme Kinetics
Chapter 6
The Michaelis-Menten Approach to Enzyme Kinetics
The Relationship between Substrate Concentration and Reaction Rate Can Be Expressed Quantitatively
- The Michaelis-Menten equation
V0 = Vmax[S]/(Km + [S]), Km = the Michaelis constant
Assumptions:
1. Early in the reaction, [P] is negligible, and the reverse reaction (P → S) can be ignored
E + S ↔ ES → E + P
 V0 is determined by the breakdown of ES to form P
V0 = k2[ES]
2. The reaction quickly achieved the steady-state in which [ES] remains constant over t

14. The Michaelis-Menten Approach to Enzyme Kinetics
Chapter 6
The Michaelis-Menten Approach to Enzyme Kinetics

15. The Michaelis-Menten Approach to Enzyme Kinetics
Chapter 6
The Michaelis-Menten Approach to Enzyme Kinetics
Kinetic Parameters Are Used to Compare Enzyme Activities
- Vmax is related to the turnover number of an enzyme, a quantity equal to the catalytic constant, k2.
V0/[Et] = turnover number = kcat
Turnover number: The number of moles of substrate that react to form product per
mole of enzyme per unit time.
For the two-step reaction: k2 = kcat
For the three-step reaction: k3 = kcat

16. Chapter 6
Enzyme Inhibition
Enzymes Are Subject to Reversible or Irreversible Inhibition:
Reversible Inhibition
- Enzyme inhibitors are molecular agents that interfere with catalysis, slowing or halting enzymatic reactions
e.g., aspirin (acetylsalicylate) inhibits the enzyme that catalyzes the first step in the synthesis of prostaglandins, compounds involved in many processes, including pain
* Competitive inhibitor: Competes with the substrate for the active site of an enzyme
* Uncompetitive inhibitor: Binds at a site distinct from the substrate active site and, unlike a competitive inhibitor, binds only to the ES complex
* Mixed inhibitor: Binds at a site distinct from the substrate active site, but it binds to either E or ES

17. Chapter 6
Enzyme Inhibition
Enzymes Are Subject to Reversible or Irreversible Inhibition: Reversible Inhibition

18. Chapter 6
Enzyme Inhibition
Enzymes Are Subject to Reversible or Irreversible Inhibition: Irreversible Inhibition
- Irreversible inhibitors are those that bind covalently with or destroy a functional group on an enzyme that is essential for the enzyme’s activity, or those that form a particularly stable noncovalent association
Reaction of chymotrypsin with diisopropylfluorophosphate (DIFP) irreversibly inhibits the enzyme. This has led to the conclusion that Ser195 is the key active-site Ser residue in chymotrypsin

19. Enzyme Inhibition Acquired immunodeficiency syndrome (AIDS)
Chapter 6
Enzyme Inhibition
Acquired immunodeficiency syndrome (AIDS)
- Specific inhibitors that selectively block the actions of enzymes unique to the human immunodeficiency virus (HIV), causing AIDS.
e.g., HIV protease: An enzyme essential to the production of new virus particles in infected cells
Active site of VX-478 complexed with HIV-1 protease

20. Enzymes, Mechanisms and Control
Chapter 7
Enzymes, Mechanisms and Control
The Behavior of Allosteric Enzymes
- Allosteric proteins are those having “other shapes” or conformations induced by the binding of allosteric effectors (modulators)
 The modulators for allosteric enzymes may be inhibitory or stimulatory
 Allosteric enzymes are either homotropic or heterotropic
- Allosteric modulators are different from uncompetitive and mixed inhibitors
 Although the inhibitors bind at a second site on the enzyme, they do not necessarily mediate conformational changes
Aspartate transcarbamoylase (ATCase)
Homotropic effects: allosteric interactions by several identical molecules bound to a protein (e.g., the binding of aspartate to ATCase)
Heterotropic effects: allosteric interactions by different substances (such as inhibitor and substrate) (e.g., Inhibition by CTP and activation by ATP)

21. Enzymes, Mechanisms and Control
Chapter 7
Enzymes, Mechanisms and Control
The Behavior of Allosteric Enzymes
Allosteic enzymes are generally larger and more complex than nonallosteric enzymes
e.g., Aspartate transcarbamoylase (12 PP chains)

22. Enzymes, Mechanisms and Control
Chapter 7
Enzymes, Mechanisms and Control
The Behavior of Allosteric Enzymes

23. Enzymes, Mechanisms and Control
Chapter 7
Enzymes, Mechanisms and Control
The Behavior of Allosteric Enzymes
The bacterial enzyme system that catalyzes the conversion of L-threonine to L-isoeucine in five steps
 The first enzyme is inhibited by isoeucine, the final product
 Heterotropic allosteric inhibition

24. Enzymes, Mechanisms and Control
Chapter 7
Enzymes, Mechanisms and Control
Control of Enzyme Activity by Phosphorylation
- For some enzymes, their activity is modulated by covalent modification
 Modifying groups include phosphoryl, adenylyl, uridylyl, methyl, and adenosine diphosphate ribosyl groups
 The modifying groups are generally linked to and removed from the regulatory enzyme by separate enzymes
 Phosphorylation is the most common type of regulatory modification (1/3 – 1/2)

25. Enzymes, Mechanisms and Control
Chapter 7
Enzymes, Mechanisms and Control
Control of Enzyme Activity by Phosphorylation
Phosphorylation of the sodium-potassium pump is involved in cycling the membrane protein
between the form that binds to sodium and the form that binds to potassium

26. Enzymes, Mechanisms and Control
Chapter 7
Enzymes, Mechanisms and Control
The Nature of the Active Site
* Chymotrypsin as a representative enzyme for the enzymatic reaction
- The side-chain reactive groups are the ones involved in the action of the enzyme
 The serine 195 and histidine 57 are required for activity
 The activity of chimotrypsin disappears when serine 195 and histidine 57 react with diisopropylphosphofluoridate and N-tosylamido-L-phenylethyl chloromethyl ketone, respectively
- Only a few residues are directly involved in the active site, but the whole molecule is necessary to provide the correct three-dimensional arrangement for those critical residues

27. Enzymes, Mechanisms and Control
Chapter 7
Enzymes, Mechanisms and Control
The Nature of the Active Site
- Chymotrypsin enhances the rate of peptide bond hydrolysis by a factor of at least 109
 Acylation: the peptide bond is cleaved and an ester linkage is formed
 Deacylation: the ester linkage is hydrolyzed and the nonacylated enzyme regenerated ①

28. Enzymes, Mechanisms and Control
Chapter 7
Enzymes, Mechanisms and Control
The Nature of the Active Site

29. Enzymes, Mechanisms and Control
Chapter 7
Enzymes, Mechanisms and Control
Coenzymes
- Cofactors are nonprotein substances that take part in enzymatic reactions and regenerated for further reaction
- Some enzymes require an additional chemical component called a cofactor – either one or more inorganic ions, or a complex organic or metalloorganic molecule called a coenzyme
Ref.) Prosthetic group: A coenzyme or metal ion that is tightly or covalently bound
to the enzyme protein
Holoenzyme: A complete, catalytically active enzyme together with its bound
coenzyme and/or metal ions
Apoenzyme (or apoprotein): The protein part of an enzyme

30. Enzymes, Mechanisms and Control
Chapter 7
Enzymes, Mechanisms and Control
Coenzymes