In the following chapters of the text, students will be introduced to a wide variety of reaction pathways and enzyme names. Although it may seem that the number of reactions is infi nite, many of these reactions are similar and occur frequently in different pathways. Recognition of the type of reaction can aid in remembering the pathways and enzyme names, thereby reducing the amount of memorization
Glycopeptide transpeptidase
Glycopeptide transpeptidase
O H
C N C
C N
C S C H
H H
C O
CH3 CH3 COO– Strained
peptide bond Penicillin
H
Ser
H O
N C
H C
C C S C O
H H N
C O
CH3 CH3 COO– Ser
OH
FIG. 6.11. The antibiotic penicillin inhibits the bacterial enzyme glycopeptide transpep- tidase. The transpeptidase is a serine protease involved in cross-linking components of bacterial cell walls and is essential for bacterial growth and survival. It normally cleaves the peptide bond between two D-alanine residues in a polypeptide. Penicillin contains a strained peptide bond within the β-lactam ring that resembles the transition state of the normal cleavage reaction, and thus, penicillin binds very readily in the enzyme active site.
As the bacterial enzyme attempts to cleave this penicillin peptide bond, penicillin becomes irreversibly covalently attached to the enzyme’s active-site serine, thereby inactivating the enzyme.
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CHAPTER 6 ■ ENZYMES AS CATALYSTS 93
C C
N GMP
AMP
Guanine
Xanthine
Xanthine oxidase
Hypoxanthine
OH N
C O– N N H
Urate C HO
N A
Xanthine oxidase
Inhibited by allopurinol
OH
N N
NH N
Hypoxanthine HC
C C C
CH
Mo = S xanthine oxidase H2O + H+
3H+,2e– HO OH
N N
NH N C
CH H
H2O + H+
3H+,2e–
O
N C
N HC
Urate
Alloxanthine-enzyme complex
Inactive enzyme Xanthine-enzyme
complex
SH O
O
HN N
NH N
Mo IV
H O
S enz Mo VI
OH N
N H C
HO C
C C
Alloxanthine (oxypurinol) N
H Allopurinol
H xanthine
oxidase OH
N HC N
C C
Xanthine B
FIG. 6.12. Allopurinol is a suicide inhibitor of xanthine oxidase. A. Xanthine oxidase catalyzes the oxidation of hypoxanthine to xanthine, and xanthine to uric acid (urate) in the pathway for degradation of purine nucleotides. B. The oxidations are performed by a molybdenum–oxo–sulfi de coordination complex in the active site that complexes with the group being oxidized. Oxygen is donated from water. The enzyme can work either as an oxidase (O2 accepts the 2e⫺ and is reduced to H2O2) or as a dehydrogenase (NAD⫹ accepts the 2e⫺ and is reduced to NADH). The fi gure only indicates that 2e⫺ are generated during the course of the reactions. C. Xanthine oxidase is able to perform the fi rst oxidation step and convert allopurinol to alloxanthine (oxypurinol). As a result, the enzyme has committed suicide; the oxypurinol remains bound in the molybdenum coor- dination sphere, where it prevents the next step of the reaction. The portion of the purine ring in green indicates the major structural differences between hypoxanthine, xanthine, and allopurinol.
required. For those interested, these reaction types are discussed online in Section A6.1 , along with the formal classifi cation of enzymes.
C L I N I CA L CO M M E N T S Diseases discussed in this chapter are summarized in Table 6.2.
Dennis V. Dennis V. survived his malathion intoxication because he had ingested only a small amount of the chemical, vomited shortly after the agent was ingested, and was rapidly treated in the emergency room. Lethal doses of oral malathion are estimated at 1 g/kg of body weight for humans. Emergency room physicians used a drug (an oxime) to reactivate the acetylcholinesterase in Dennis before an aged irreversible complex formed. They also used intravenous atropine, an anticholinergic (antimuscarinic) agent, to antagonize the action of the excessive amounts of acetylcholine accumulating in cholinergic receptors throughout his body.
After several days of intravenous therapy, the signs and symptoms of acetylcho- line excess abated and therapy was slowly withdrawn. Dennis made an uneventful recovery.
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Table 6.2 Diseases Discussed in Chapter 6 Disorder or Condition
Genetic or
Environmental Comments
Malathion poisoning Environmental Inhibition of acetylcholinesterase at neuromus- cular junctions. This leads to acetylcholine accumulation at the junction and overstimu- lation of the autonomic nervous system.
Gout Both Accumulation of uric acid in blood, leading to
precipitation in joints, accompanied by se- vere pain and discomfort.
Thiamin defi ciency (beriberi heart disease)
Environmental Leads to lack of energy production due to reduced activity of key enzymes and can lead to the Wernicke-Korsakoff syndrome.
Brought about by alcoholism, as manifest by a poor diet, and ethanol inhibition of thiamine transport through the intestinal mucosa.
Lotta T. Within several days of starting allopurinol therapy, Ms. T.’s serum uric acid level began to fall. Several weeks later, the level in her blood was normal. However, while Lotta was adapting to allopurinol therapy, she experienced a mild gout attack, which was treated with a low dose of colchicine (see Chapter 8).
Al M. Al M. was admitted to the hospital after intravenous thiamine was initiated at a dose of 100 mg/day (compared to an RDA of 1.4 mg/day). His congestive heart failure was believed to be the result, in part, of the cardio- myopathy (heart muscle dysfunction) of acute thiamine defi ciency known as beri- beri heart disease. This cardiac dysfunction and the peripheral nerve dysfunction that result from this nutritional defi ciency usually respond to thiamine replacement.
However, an alcoholic cardiomyopathy can also occur in well-nourished patients with adequate thiamine levels. Exactly how ethanol, or its toxic metabolite acetalde- hyde, causes alcoholic cardiomyopathy in the absence of thiamine defi ciency is not completely understood.
At low concentrations of ethanol, liver alcohol dehydrogenase is the major route of ethanol oxidation to form acetaldehyde, a highly toxic chemical.
Acetaldehyde not only damages the liver, it can enter the blood and potentially damage the heart and other tissues. At low ethanol intakes, much of the acetaldehyde produced is safely oxidized to acetate in the liver by aldehyde de- hydrogenases.
Once malathion is ingested, the liver converts it to the toxic reactive compound malaoxon by converting the phosphate–sulfur double bond to a phosphate– oxygen double bond. Malaoxon then binds to the active site of acetylcholinesterase and reacts to form the covalent intermediate. Unlike the complex formed between DFP and ace- tylcholinesterase, this initial acyl–enzyme intermediate is reversible. However, with time, the enzyme inhibitor complex “ages” (dealkylation of the inhibitor and enzyme modifi cation) to form an irreversible complex.
H CH3O P S C
CH2 OCH3
OCH2CH3 C
O
OCH2CH3 C O S
Malathion
H CH3O P S C
CH2 OCH3
OCH2CH3 C
O
OCH2CH3 C O O
Malaoxon
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CHAPTER 6 ■ ENZYMES AS CATALYSTS 95
1. A patient was born with a congenital mutation in an en- zyme severely affecting its ability to bind an activation- transfer coenzyme. As a consequence, which one of the following would you expect to occur?
A. The enzyme would be unable to bind the substrate of the reaction.
B. The enzyme would be unable to form the transition state complex.
C. The enzyme would normally use a different activa- tion-transfer coenzyme.
D. The enzyme would normally substitute the func- tional group of an active site amino acid residue for the coenzyme.
E. The reaction could be carried out by the free coen- zyme, provided the diet carried an adequate amount of its vitamin precursor.
2. An individual had a congenital mutation in glucokinase in which a proline was substituted for a leucine on a surface helix far from the active site, but within the hinge region of the actin fold. This mutation would be expected to do which one of the following?
A. Probably affect the binding of ATP or a subsequent step in the reaction sequence.
B. Have no effect on the rate of the reaction because it is not in the active site.
C. Have no effect on the rate of the reaction because proline and leucine are both nonpolar amino acids.
D. Have no effect on the number of substrate molecules reaching the transition state.
E. Probably cause the reaction to proceed through an alternate mechanism.
3. A patient developed a bacterial overgrowth in his intestine that decreased the pH of the luminal contents from their normal pH of about 6.5 down to 5.5. This decrease of pH is likely to lead to which one of the following?
A. Inhibit intestinal enzymes dependent on an active site lysine for binding substrate.
B. Have little effect on intestinal hydrolases.
C. Denature proteins reaching the intestine with their native structure intact.
D. Disrupt hydrogen bonding essential for maintenance of tertiary structure.
E. Inhibit intestinal enzymes dependent on histidine for acid-base catalysis.
4. Enzymes accelerate reaction rates due to which one of the following?
A. Increase the effective concentration of the substrate.
B. Reduce the frequency of collisions between the sub- strate molecules.
C. Lower the energy of activation required to reach a transition state.
D. Increase the free energy level of the fi nal state of the reaction.
E. Decrease the free energy level of the initial state of the reaction.
5. The enzyme alcohol dehydrogenase catalyzes the conver- sion of ethanol to acetaldehyde. Which one of the follow- ing occurs during the course of this reaction?
A. The substrate, ethanol, is oxidized.
B. An active site serine residue is oxidized.
C. The coenzyme, NAD, is oxidized.
D. An active site zinc atom is oxidized.
E. Thiamine pyrophosphate is oxidized.
R E V I E W Q U E ST I O N S - C H A P T E R 6
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96
7 Regulation of Enzymes
K E Y P O I N T S
■ Enzyme activity is regulated to refl ect the physiological state of the organism.
■ The rate of an enzyme-catalyzed reaction is dependent on substrate concentration and can be repre- sented mathematically by the Michaelis-Menten equation.
■ Allosteric activators or inhibitors are compounds that bind at sites other than the active catalytic site and regulate the enzyme through conformational changes affecting the catalytic site.
■ A number of different mechanisms are available to regulate enzyme activity. These include:
■ Feedback inhibition, which often occurs at the fi rst committed step of a metabolic pathway