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Berg JM, Tymoczko JL, Stryer L. Biochemistry. 5th edition. New York: W H Freeman; 2002.

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Biochemistry. 5th edition.

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Section 8.5Enzymes Can Be Inhibited by Specific Molecules

The activity of many enzymes can be inhibited by the binding of specific small molecules and ions. This means of inhibiting enzyme activity serves as a major control mechanism in biological systems. The regulation of allosteric enzymes typifies this type of control. In addition, many drugs and toxic agents act by inhibiting enzymes. Inhibition by particular chemicals can be a source of insight into the mechanism of enzyme action: specific inhibitors can often be used to identify residues critical for catalysis. The value of transition-state analogs as potent inhibitors will be discussed shortly.

Enzyme inhibition can be either reversible or irreversible. An irreversible inhibitor dissociates very slowly from its target enzyme because it has become tightly bound to the enzyme, either covalently or noncovalently. Some irreversible inhibitors are important drugs. Penicillin acts by covalently modifying the enzyme transpeptidase, thereby preventing the synthesis of bacterial cell walls and thus killing the bacteria (Section 8.5.5). Aspirin acts by covalently modifying the enzyme cyclooxygenase, reducing the synthesis of inflammatory signals.

Reversible inhibition, in contrast with irreversible inhibition, is characterized by a rapid dissociation of the enzyme-inhibitor complex. In competitive inhibition, an enzyme can bind substrate (forming an ES complex) or inhibitor (EI) but not both (ESI). The competitive inhibitor resembles the substrate and binds to the active site of the enzyme (Figure 8.15). The substrate is thereby prevented from binding to the same active site. A competitive inhibitor diminishes the rate of catalysis by reducing the proportion of enzyme molecules bound to a substrate. At any given inhibitor concentration, competitive inhibition can be relieved by increasing the substrate concentration. Under these conditions, the substrate “outcompetes” the inhibitor for the active site. Methotrexate is a structural analog of tetrahydrofolate, a coenzyme for the enzyme dihydrofolate reductase, which plays a role in the biosynthesis of purines and pyrimidines (Figure 8.16). It binds to dihydrofolate reductase 1000-fold more tightly than the natural substrate and inhibits nucleotide base synthesis. It is used to treat cancer.

Figure 8.15. Distinction between a Competitive and a Noncompetitive Inhibitor.

Figure 8.15

Distinction between a Competitive and a Noncompetitive Inhibitor. (Top) enzyme-substrate complex; (middle) a competitive inhibitor binds at the active site and thus prevents the substrate from binding; (bottom) a noncompetitive inhibitor does not prevent (more...)

Figure 8.16. Enzyme Inhibitors.

Figure 8.16

Enzyme Inhibitors. The cofactor tetrahydrofolate and its structural analog methotrexate. Regions with structural differences are shown in red.

In noncompetitive inhibition, which also is reversible, the inhibitor and substrate can bind simultaneously to an enzyme molecule at different binding sites (see Figure 8.16). A noncompetitive inhibitor acts by decreasing the turnover number rather than by diminishing the proportion of enzyme molecules that are bound to substrate. Noncompetitive inhibition, in contrast with competitive inhibition, cannot be overcome by increasing the substrate concentration. A more complex pattern, called mixed inhibition, is produced when a single inhibitor both hinders the binding of substrate and decreases the turnover number of the enzyme.

8.5.1. Competitive and Noncompetitive Inhibition Are Kinetically Distinguishable

How can we determine whether a reversible inhibitor acts by competitive or noncompetitive inhibition? Let us consider only enzymes that exhibit Michaelis-Menten kinetics. Measurements of the rates of catalysis at different concentrations of substrate and inhibitor serve to distinguish the two types of inhibition. In competitive inhibition, the inhibitor competes with the substrate for the active site. The dissociation constant for the inhibitor is given by

Image ch8e45.jpg

Because increasing the amount of substrate can overcome the inhibition, Vmax can be attained in the presence of a competitive inhibitor (Figure 8.17). The hallmark of competitive inhibition is that it can be overcome by a sufficiently high concentration of substrate. However, the apparent value of KM is altered; the effect of a competitive inhibitor is to increase the apparent value of KM. This new value of KM, called KappM , is numerically equal to

Image ch8e46.jpg
where [I] is the concentration of inhibitor and Ki is the dissociation constant for the enzyme-inhibitor complex. As the value of [I] increases, the value of KappM increases (see Figure 8.17). In the presence of a competitive inhibitor, an enzyme will have the same Vmax as in the absence of an inhibitor.

Figure 8.17. Kinetics of a Competitive Inhibitor.

Figure 8.17

Kinetics of a Competitive Inhibitor. As the concentration of a competitive inhibitor increases, higher concentrations of substrate are required to attain a particular reaction velocity. The reaction pathway suggests how sufficiently high concentrations (more...)

In noncompetitive inhibition (Figure 8.18), substrate can still bind to the enzyme-inhibitor complex. However, the enzyme-inhibitor-substrate complex does not proceed to form product. The value of Vmax is decreased to a new value called V appmax while the value of KM is unchanged. Why is Vmax lowered while KM remains unchanged? In essence, the inhibitor simply lowers the concentration of functional enzyme. The remaining enzyme behaves like a more dilute solution of enzyme; Vmax is lower, but KM is the same. Noncompetitive inhibition cannot be overcome by increasing the substrate concentration.

Figure 8.18. Kinetics of a Noncompetitive Inhibitor.

Figure 8.18

Kinetics of a Noncompetitive Inhibitor. The reaction pathway shows that the inhibitor binds both to free enzyme and to enzyme complex. Consequently, Vmax cannot be attained, even at high substrate concentrations.

8.5.2. Irreversible Inhibitors Can Be Used to Map the Active Site

In Chapter 9, we will examine the chemical details of how enzymes function. The first step in obtaining the chemical mechanism of an enzyme is to determine what functional groups are required for enzyme activity. How can we ascertain these functional groups? X-ray crystallography (Section 4.5.2) of the enzyme bound to its substrate provides one approach. Irreversible inhibitors that covalently bond to the enzyme provide an alternative and often complementary means for elucidating functional groups at the enzyme active site because they modify the functional groups, which can then be identified. Irreversible inhibitors can be divided into three categories: group-specific reagents, substrate analogs, and suicide inhibitors.

Group-specific reagents react with specific R groups of amino acids. Two examples of group-specific reagents are diisopropylphosphofluoridate (DIPF; Figure 8.19) and iodoacetamide (Figure 8.20). DIPF modifies only 1 of the 28 serine residues in the proteolytic enzyme chymotrypsin, implying that this serine residue is especially reactive. As we will see in Chapter 9, it is indeed the case that this serine residue is at the active site. DIPF also revealed a reactive serine residue in acetylcholinesterase, an enzyme important in the transmission of nerve impulses (see Figure 8.19). Thus, DIPF and similar compounds that bind and inactivate acetylcholinesterase are potent nerve gases.

Figure 8.19. Enzyme Inhibition by Diisopropylphosphofluoridate (DIPF), a Group-Specific Reagent.

Figure 8.19

Enzyme Inhibition by Diisopropylphosphofluoridate (DIPF), a Group-Specific Reagent. DIPF can inhibit an enzyme by covalently modifying a crucial serine residue (Section 9.1.1).

Figure 8.20. Enzyme Inactivation by Iodoacetamide, a Group-Specific Reagent.

Figure 8.20

Enzyme Inactivation by Iodoacetamide, a Group-Specific Reagent. Iodoacetamide can inactivate an enzyme by reacting with a critical cysteine residue.

Affinity labels are molecules that are structurally similar to the substrate for the enzyme that covalently modify active site residues. They are thus more specific for the enzyme active site than are group-specific reagents. Tosyl-l-phenylalanine chloromethyl ketone (TPCK) is a substrate analog for chymotrypsin (Figure 8.21). TPCK binds at the active site; and then reacts irreversibly with a histidine residue at that site, inhibiting the enzyme. The compound 3-bromoacetol is an affinity label for the enzyme triose phosphate isomerase (TIM). It mimics the normal substrate, dihydroxyacetone phosphate, by binding at the active site; then it covalently modifies the enzyme such that the enzyme is irreversibly inhibited (Figure 8.22).

Figure 8.21. Affinity Labeling.

Figure 8.21

Affinity Labeling. (A) Tosyl-l-phenylalanine chloromethyl ketone (TPCK) is a reactive analog of the normal substrate for the enzyme chymotrypsin. (B) TPCK binds at the active site of chymotrypsin and modifies an essential histidine residue.

Figure 8.22. Bromoacetol Phosphate, an Affinity Label for Triose Phosphate Isomerase (TIM).

Figure 8.22

Bromoacetol Phosphate, an Affinity Label for Triose Phosphate Isomerase (TIM). Bromoacetol phosphate, an analog of dihydroxyacetone phosphate, binds at the active site of the enzyme and covalently modifies a glutamic acid residue required for enzyme activity. (more...)

Suicide inhibitors, or mechanism-based inhibitors are modified substrates that provide the most specific means to modify an enzyme active site. The inhibitor binds to the enzyme as a substrate and is initially processed by the normal catalytic mechanism. The mechanism of catalysis then generates a chemically reactive intermediate that inactivates the enzyme through covalent modification. The fact that the enzyme participates in its own irreversible inhibition strongly suggests that the covalently modified group on the enzyme is catalytically vital. One example of such an inhibitor is N,N-dimethylpropargylamine. A flavin prosthetic group of monoamine oxidase (MAO) oxidizes the N,N-dimethylpropargylamine, which in turn inactivates the enzyme by covalently modifying the flavin prosthetic group by alkylating N-5 (Figure 8.23). Monoamine oxidase deaminates neurotransmitters such as dopamine and serotonin, lowering their levels in the brain. Parkinson disease is associated with low levels of dopamine, and depression is associated with low levels of serotonin. The drug (-)deprenyl, which is used to treat Parkinson disease and depression, is a suicide inhibitor of monoamine oxidase.

Figure 8.23. Mechanism-Based (Suicide) Inhibition.

Figure 8.23

Mechanism-Based (Suicide) Inhibition. Monoamine oxidase, an enzyme important for neurotransmitter synthesis, requires the cofactor FAD (flavin adenine dinucleotide). N,N-Dimethylpropargylamine inhibits monoamine oxidase by covalently modifying the flavin (more...)

8.5.3. Transition-State Analogs Are Potent Inhibitors of Enzymes

We turn now to compounds that provide the most intimate views of the catalytic process itself. Linus Pauling proposed in 1948 that compounds resembling the transition state of a catalyzed reaction should be very effective inhibitors of enzymes. These mimics are called transition-state analogs. The inhibition of proline racemase is an instructive example. The racemization of proline proceeds through a transition state in which the tetrahedral α- carbon atom has become trigonal by loss of a proton (Figure 8.24). In the trigonal form, all three bonds are in the same plane; Cα also carries a net negative charge. This symmetric carbanion can be reprotonated on one side to give the l isomer or on the other side to give the d isomer. This picture is supported by the finding that the inhibitor pyrrole 2-carboxylate binds to the racemase 160 times as tightly as does proline. The α-carbon atom of this inhibitor, like that of the transition state, is trigonal. An analog that also carries a negative charge on Cα would be expected to bind even more tightly. In general, synthesizing compounds that more closely resemble the transition state than the substrate itself can produce highly potent and specific inhibitors of enzymes. The inhibitory power of transition-state analogs underscores the essence of catalysis: selective binding of the transition state.

Image ch8fu14.jpg

Figure 8.24. Inhibition by Transition State Analogs.

Figure 8.24

Inhibition by Transition State Analogs. (A) The isomerization of l-proline to d-proline by proline racemase, a bacterial enzyme, proceeds through a planar transition state in which the α carbon is trigonal rather than tetrahedral. (B) Pyrrole (more...)

8.5.4. Catalytic Antibodies Demonstrate the Importance of Selective Binding of the Transition State to Enzymatic Activity

Antibodies that recognize transition states should function as catalysts, if our understanding of the importance of the transition state to catalysis is correct. The preparation of an antibody that catalyzes the insertion of a metal ion into a porphyrin nicely illustrates the validity of this approach. Ferrochelatase, the final enzyme in the biosynthetic pathway for the production of heme, catalyzes the insertion of Fe2+ into protoporphyrin IX. The nearly planar porphyrin must be bent for iron to enter. The recently determined crystal structure of the ferrochelatase bound to a substrate analog confirms that the enzyme does indeed bend one of the pyrole rings, distorting it 36 degrees to insert the iron.

The problem was to find a transition-state analog for this metallation reaction that could be used as an antigen (immunogen) to generate an antibody. The solution came from the results of studies showing that an alkylated porphyrin, N-methylprotoporphyrin, is a potent inhibitor of ferrochelatase. This compound resembles the transition state because N-alkylation forces the porphyrin to be bent. Moreover, it was known that N-alkylporphyrins chelate metal ions 104 times as fast as their unalkylated counterparts do. Bending increases the exposure of the pyrrole nitrogen lone pairs of electrons to solvent, which facilitates metal in binding.

An antibody catalyst was produced with the use of an N-alkylporphyrin as the immunogen. The resulting antibody presumably distorts a planar porphyrin (Figure 8.25) to facilitate the entry of a metal. On average, an antibody molecule metallated 80 porphyrin molecules per hour, a rate only 10-fold less than that of ferrochelatase and 2500-fold faster than the uncatalyzed reaction. Catalytic antibodies (abzymes) can indeed be produced by using transition-state analogs as antigens. Antibodies catalyzing many other kinds of chemical reactions—exemplified by ester and amide hydrolysis, amide-bond formation, transesterification, photoinduced cleavage, photoinduced dimerization, decarboxylation, and oxidization—have been produced with the use of similar strategies. The results of studies with transition-state analogs provide strong evidence that enzymes can function complementary in structure to the transition state. The power of transition-state analogs is now evident: (1) they are sources of insight into catalytic mechanisms, (2) they can serve as potent and specific inhibitors of enzymes, and (3) they can be used as immunogens to generate a wide range of novel catalysts.

Figure 8.25. Use of Transition-State Analogs to Generate Catalytic Antibodies.

Figure 8.25

Use of Transition-State Analogs to Generate Catalytic Antibodies. The insertion of a metal ion into a porphyrin by ferrochelatase proceeds through a transition state in which the porphyrin is bent. N-Methylmesoporphyrin, a bent porphyrin that resembles (more...)

8.5.5. Penicillin Irreversibly Inactivates a Key Enzyme in Bacterial Cell-Wall Synthesis

Image caduceus.jpg Penicillin, the first antibiotic discovered, consists of a thiazolidine ring fused to a β-lactam ring, to which a variable R group is attached by a peptide bond (Figure 8.26A). In benzyl penicillin, for example, R is a benzyl group (Figure 8.26B). This structure can undergo a variety of rearrangements, and, in particular, the β-lactam ring is very labile. Indeed, this instability is closely tied to the antibiotic action of penicillin, as will be evident shortly.

Figure 8.26. Structure of Penicillin.

Figure 8.26

Structure of Penicillin. The reactive site of penicillin is the peptide bond of its β-lactam ring. (A) Structural formula of penicillin. (B) Representation of benzyl penicillin.

How does penicillin inhibit bacterial growth? In 1957, Joshua Lederberg showed that bacteria ordinarily susceptible to penicillin could be grown in its presence if a hypertonic medium were used. The organisms obtained in this way, called protoplasts, are devoid of a cell wall and consequently lyse when transferred to a normal medium. Hence, penicillin was inferred to interfere with the synthesis of the bacterial cell wall. The cell-wall macromolecule, called a peptidoglycan, consists of linear polysaccharide chains that are cross-linked by short peptides (Figure 8.27). The enormous bag-shaped peptidoglycan confers mechanical support and prevents bacteria from bursting in response to their high internal osmotic pressure.

Figure 8.27. Schematic Representation of the Peptidoglycan in Staphylococcus aureus.

Figure 8.27

Schematic Representation of the Peptidoglycan in Staphylococcus aureus. The sugars are shown in yellow, the tetrapeptides in red, and the pentaglycine bridges in blue. The cell wall is a single, enormous, bag-shaped macromolecule because of extensive (more...)

In 1965, James Park and Jack Strominger independently deduced that penicillin blocks the last step in cell-wall synthesis—namely, the crosslinking of different peptidoglycan strands. In the formation of the cell wall of Staphylococcus aureus, the amino group at one end of a pentaglycine chain attacks the peptide bond between two d-alanine residues in another peptide unit (Figure 8.28). A peptide bond is formed between glycine and one of the d-alanine residues; the other d-alanine residue is released. This cross-linking reaction is catalyzed by glycopeptide transpeptidase. Bacterial cell walls are unique in containing d amino acids, which form cross-links by a mechanism different from that used to synthesize proteins.

Figure 8.28. Formation of Cross-Links in S. aureus Peptidoglycan.

Figure 8.28

Formation of Cross-Links in S. aureus Peptidoglycan. The terminal amino group of the pentaglycine bridge in the cell wall attacks the peptide bond between two d-alanine residues to form a cross-link.

Penicillin inhibits the cross-linking transpeptidase by the Trojan horse stratagem. The transpeptidase normally forms an acyl intermediate with the penultimate d-alanine residue of the d-Ala-d-Ala peptide (Figure 8.29). This covalent acyl-enzyme intermediate then reacts with the amino group of the terminal glycine in another peptide to form the cross-link. Penicillin is welcomed into the active site of the transpeptidase because it mimics the d-Ala-d-Ala moiety of the normal substrate (Figure 8.30). Bound penicillin then forms a covalent bond with a serine residue at the active site of the enzyme. This penicilloyl-enzyme does not react further. Hence, the transpeptidase is irreversibly inhibited and cell-wall synthesis cannot take place.

Figure 8.29. Transpeptidation Reaction.

Figure 8.29

Transpeptidation Reaction. An acyl-enzyme intermediate is formed in the transpeptidation reaction leading to cross-link formation.

Figure 8.30. Conformations of Penicillin and a Normal Substrate.

Figure 8.30

Conformations of Penicillin and a Normal Substrate. The conformation of penicillin in the vicinity of its reactive peptide bond (A) resembles the postulated conformation of the transition state of R-d-Ala-d-Ala (B) in the transpeptidation reaction. [After (more...)

Why is penicillin such an effective inhibitor of the transpeptidase? The highly strained, four-membered β-lactam ring of penicillin makes it especially reactive. On binding to the transpeptidase, the serine residue at the active site attacks the carbonyl carbon atom of the lactam ring to form the penicilloyl-serine derivative (Figure 8.31). Because the peptidase participates in its own inactivation, penicillin acts as a suicide inhibitor.

Figure 8.31. Formation of a Penicilloyl-Enzyme Complex.

Figure 8.31

Formation of a Penicilloyl-Enzyme Complex. Penicillin reacts with the transpeptidase to form an inactive complex, which is indefinitely stable.

By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2002, W. H. Freeman and Company.
Bookshelf ID: NBK22530