BIOCHEM DISCUSSION 3

Alright, now we're going to talk briefly about some enzymatic mechanisms and inhibitors.

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Biochemistry: A Short Course Fourth Edition

CHAPTER 8 Mechanisms and Inhibitors

Tymoczko • Berg • Gatto • Stryer

© 2019 W. H. Freeman and Company.

8.1: First, we're going to talk about how a few basic catalytic strategies are used by many enzymes, and that’s actually a motif in nature, generally speaking. Once nature has solved the problem, she reuses that solution over and over again. 8.2: And we're also going to be talking about how enzyme activity can be modulated by temperature, pH, and inhibitory molecules. 8.3: I am skipping the section on chymotrypsin, but I encourage you to read it in the book. It is fascinating but not necessary for what we are going to be talking about.

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Chapter 8 Outline

8.1 A Few Basic Catalytic Strategies Are Used by Many Enzymes

8.2 Enzyme Activity Can Be Modulated by Temperature, pH, and Inhibitory Molecules

8.3 Chymotrypsin Illustrates Basic Principles of Catalysis and Inhibition

Here are four common catalytic strategies (there are many more, of course, but): 1. One of them is covalent catalysis, where things are briefly covalently linked to each other in the active site. 2. Then there is general acid-base catalysis, which is just shuttling around of protons. 3. Metal ion catalysis, where they use metal ions to perform the catalytic reactions. 4. And catalysis by approximation and orientation is the lining up of the substrates in a special way so that they will react.

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8.1 A Few Basic Catalytic Strategies Are Used by Many Enzymes (1/2)

Common catalytic strategies include: 1. Covalent catalysis: The active site contains a nucleophile

that is briefly covalently modified. 2. General acid–base catalysis: A molecule other than water

donates or accepts a proton. 3. Metal ion catalysis: Metal ions function in a number of

ways, including serving as an electrophilic catalyst. 4. Catalysis by approximation and orientation: The enzyme

brings two substrates together in an orientation that facilitates catalysis.

Temperature affects the rate of enzyme-catalyzed reactions, much as it affects the rate of regular chemical reactions - but, it has some particular effects with enzyme- catalyzed reactions. And most enzymes also have an optimal pH that they're functional at.

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Section 8.2 Enzyme Activity Can Be Modulated by Temperature, pH, and Inhibitory Molecules

Learning objective 6: List environmental factors that affect enzyme activity and describe how these factors exert their effects on enzymes.

• Temperature Enhances the Rate of Enzyme-Catalyzed Reactions

• Most Enzymes Have an Optimal pH

Figure 8.1 The effect of heat on enzyme activity. The enzyme tyrosinase, which is part of the pathway that synthesizes the pigment that results in dark fur, has a low tolerance for heat in Siamese cats. It is inactive at normal body temperatures but functional at slightly lower temperatures. The extremities of a Siamese cat are cool enough for tyrosinase to gain function and produce pigment. [Photograph: Jane Burton/Getty.]

An excellent example of a heat-sensitive enzyme would be the tyrosinase found in the Siamese cat. At normal body temperatures, it’s non-functional - but, at slightly smaller temperatures where the cat is cooler, it is functional and is therefore able to produce pigment. And that's why you see pigment in the nose and the ears, tail, and the feet of the cat - because in those places, the cat is a little cooler and the tyrosinase is able to function. One common reason that enzymes might not be able to function at higher temperature is because they denature and lose their structure at higher temperatures.

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Graph Model of the Effect of Heat on Enzyme Activity

Figure 8.4 The pH dependence of the activity of the enzymes pepsin and chymotrypsin. Chymotrypsin and pepsin have different optimal pH values. The optimal pH for pepsin is noteworthy. Most proteins would be denatured at this acidic pH.

And, here's a probably rather idealized graph of the activity of pepsin and chymotrypsin. You can see, on the left, that pepsin is most active at a pH between 1 and 2. Well, that's where it does its work - in the stomach, where there's a lot of hydrochloric acid and the pH is very low - so the protein has become optimized so that it can function at that specific pH. Most enzymes actually function near the body pH, which is around {7.4}, because that's the pH that they are normally found in. But modulating pH is actually a way that your body can control the activity of certain enzymes.

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Graph of the pH Dependence of the Activity of the Enzymes Pepsin and Chymotrypsin

A third way to control enzymes is with inhibitory molecules, and this is one of the most sensitive and prevalent methods of inhibiting an enzymatic reaction. So we're talking about competitive inhibition, uncompetitive inhibition, and noncompetitive inhibition; and I hate these terms uncompetitive and noncompetitive - they really don't make any sense to me - but I'll show you what they look like.

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Enzymes Can Be Inhibited by Specific Molecules

There are three common types of reversible inhibition: 1. Competitive inhibition: The inhibitor is structurally similar

to the substrate and can bind to the active site, preventing the actual substrate from binding.

2. Uncompetitive inhibition: The inhibitor binds only to the enzyme–substrate complex.

3. Noncompetitive inhibition: The inhibitor binds either the enzyme or enzyme–substrate complex.

Figure 8.6 Reversible inhibitors. (A) Substrate binds to an enzyme’s active site to form an enzyme–substrate complex. (B) A competitive inhibitor binds at the active site and thus prevents the substrate from binding. (C) An uncompetitive inhibitor binds only to the enzyme–substrate complex. (D) A noncompetitive inhibitor does not prevent the substrate from binding.

(A) At the top here, we have the enzyme-substrate complex. The substrate fits into the active site of the enzyme. (B) A competitive inhibitor also fits into the active site of the enzyme, and therefore competes with the substrate. When the competitive inhibitor is bound, the catalyzed reaction cannot take place, and therefore the rate of the overall reaction is slowed down. In (C) we see an uncompetitive inhibitor. It doesn’t bind to the active site, but only binds to the enzyme-substrate complex, after the substrate has already bound, preventing the reaction from proceeding. (D) And then the fourth, the noncompetitive inhibitor, binds away from the active site, whether the substrate is bound or not. Its binding affects the overall shape of the enzyme, such as altering the active site itself and thus prevents it from performing its usual functions. Binding to an external place on the enzyme and changing its conformation is an example of allosteric inhibition. “Allo-” means other, indicating not in the active site and “-steric” is a term that we often use to indicate when things are blocking each other.

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Competitive, Uncompetitive, and Noncompetitive inhibitors

Penicillin is an excellent example of an inhibitor molecule.

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Clinical Insight: Penicillin (1/3)

CLINICAL INSIGHT Penicillin Irreversibly Inactivates a Key Enzyme in Bacterial Cell-Wall Synthesis

• Penicillin is an antibiotic that consists of a thiazolidine ring fused to a very reactive β-lactam ring.

• Penicillin inhibits the formation of cell walls in certain bacteria such as S. aureus.

Figure 8.15 The structure of penicillin. The reactive site of penicillin is the peptide bond of its b-lactam ring.

It has a structure that is very similar to the substrate of an important protein for creating the cell wall in bacteria. And therefore, it fits into the active site.

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Diagram of the Structure of Penicillin

Figure 8.19 The formation of a penicilloyl-enzyme complex. Penicillin reacts with the transpeptidase to form an inactive complex, which is indefinitely stable.

When it gets to the active site, it binds to one of the residues there - the serine residue - and creates an irreversible complex, and therefore permanently blocks that site.

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Diagram of the Formation of a Penicilloyl-enzyme Complex

• When penicillin binds to the peptidase, a serine residue at the active site attacks the carbonyl carbon of the lactam ring as if penicillin were a substrate.

• A penicilloyl-serine derivative is formed that is inactive and very stable.