Drug development often involves more than the kinetic evaluation of the interaction of inhibitors with the target enzyme. Drugs are acted upon by enzymes present in the patient or pathogen, a process termed drug metabolism. For example, penicillin and other -lactam antibiotics block cell wall synthesis in bacteria by irreversibly poisoning the enzyme alanyl alanine carboxypeptidase-transpeptidase. Many bacteria, however, produce -lactamases that hydrolyze the critical -lactam function in penicillin and related drugs. One strategy for overcoming the resulting antibiotic resistance is to simultaneously administer a -lactamase inhibitor and a -lactam antibiotic.
Metabolic transformation is also required to convert an inactive drug precursor, or prodrug, into its biologically active form (Chapter 53). 2'-Deoxy-5-fluorouridylic acid, a potent inhibitor of thymidylate synthase, a common target of cancer chemotherapy, is produced from 5-fluorouracil via a series of enzymatic transformations catalyzed by a phosphoribosyl transferase and the enzymes of the deoxyribonucleoside salvage pathway (Chapter 33). Effective design and administration of prodrugs requires knowledge of the kinetics and mechanisms of the enzymes responsible for transforming them into their biologically active forms.
SUMMARY
The study of enzyme kinetics—the factors that affect the rates of enzyme-catalyzed
reactions—reveals the individual steps by which enzymes transform substrates into products.
G, the overall change in free energy for a reaction, is independent of reaction mechanism and provides no information concerning rates of reactions.
Enzymes do not affect Keq. Keq, a ratio of reaction rate constants, may be calculated from the concentrations of substrates and products at equilibrium or from the ratio k1/k– 1.
Reactions proceed via transition states in which GF is the activation energy. Temperature, hydrogen ion concentration, enzyme concentration, substrate concentration, and inhibitors all affect the rates of enzyme-catalyzed reactions.
Measurement of the rate of an enzyme-catalyzed reaction generally employs initial rate conditions, for which the essential absence of product precludes the reverse reaction.
Linear forms of the Michaelis-Menten equation simplify determination of Km and Vmax.
A linear form of the Hill equation is used to evaluate the cooperative substrate-binding kinetics exhibited by some multimeric enzymes. The slope n, the Hill coefficient, reflects the number, nature, and strength of the interactions of the substrate-binding sites. A value of n greater than 1 indicates positive cooperativity.
The effects of simple competitive inhibitors, which typically resemble substrates, are overcome by raising the concentration of the substrate. Simple noncompetitive inhibitors lower Vmax but do not affect Km.
For simple competitive and noncompetitive inhibitors, the inhibitory constant Ki is equal to the equilibrium dissociation constant for the relevant enzyme-inhibitor complex. A simpler and less rigorous term for evaluating the effectiveness of an inhibitor is IC5 0, the concentration of inhibitor that produces 50% inhibition under the particular circumstances of the experiment.
Substrates may add in a random order (either substrate may combine first with the enzyme) or in a compulsory order (substrate A must bind before substrate B).
In ping-pong reactions, one or more products are released from the enzyme before all the substrates have been added.
Applied enzyme kinetics facilitates the identification and characterization of drugs that selectively inhibit specific enzymes. Enzyme kinetics thus plays a central and critical role in drug discovery, in comparative pharmacodynamics, and in determining the mode of action of drugs.
REFERENCES
Dixon M: The determination of enzyme inhibitor constants. Biochem J 1953;55:170. [PMID: 13093635]
Dixon M: The graphical determination of K m and K i. Biochem J 1972;129:197. [PMID: 4630451]
Fersht A: Structure and Mechanism in Protein Science: A Guide to Enzyme Catalysis and Protein Folding.
Freeman, 1999.
Fraser CM, Rappuoli R: Application of microbial genomic science to advanced therapeutics. Annu Rev Med 2005;56:459. [PMID: 15660522]
Henderson PJF: A linear equation that describes the steady-state kinetics of enzymes and subcellular particles interacting with tightly bound inhibitors. Biochem J 1972;127:321. [PMID: 4263188]
Schramm, VL: Enzymatic transition-state theory and transition-state analogue design. J Biol Chem 2007;282:28297. [PMID: 17690091]
Schultz AR: Enzyme Kinetics: From Diastase to Multi-enzyme Systems. Cambridge University Press, 1994.
Segel IH: Enzyme Kinetics. Wiley Interscience, 1975.
Wlodawer A: Rational approach to AIDS drug design through structural biology. Annu Rev Med 2002;53:595. [PMID: 11818491]
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Harper's Illustrated Biochemistry, 28e > Chapter 9. Enzymes: Regulation of Activities >
BIOMEDICAL IMPORTANCE
The 19th-century physiologist Claude Bernard enunciated the conceptual basis for metabolic regulation. He observed that living organisms respond in ways that are both quantitatively and temporally appropriate to permit them to survive the multiple challenges posed by changes in their external and internal
environments. Walter Cannon subsequently coined the term "homeostasis" to describe the ability of animals to maintain a constant intracellular environment despite changes in their external environment. We now know that organisms respond to changes in their external and internal environment by balanced,
coordinated adjustments in the rates of specific metabolic reactions. Perturbations of the sensor-response machinery responsible for maintaining homeostatic balance can be deleterious to human health. Cancer, diabetes, cystic fibrosis, and Alzheimer's disease, for example, are all characterized by regulatory dysfunctions triggered by pathogenic agents or genetic mutations. Many oncogenic viruses elaborate protein-tyrosine kinases that modify the regulatory events that control patterns of gene expression, contributing to the initiation and progression of cancer. The toxin from Vibrio cholerae, the causative agent of cholera, disables sensor-response pathways in intestinal epithelial cells by ADP-ribosylating the GTP- binding proteins (G-proteins) that link cell surface receptors to adenylyl cyclase. The consequent activation of the cyclase leads to the unrestricted flow of water into the intestines, resulting in massive diarrhea and dehydration. Yersinia pestis, the causative agent of plague, elaborates a protein-tyrosine phosphatase that hydrolyzes phosphoryl groups on key cytoskeletal proteins. Dysfunctions in the proteolytic systems
responsible for the degradation of defective or abnormal proteins are believed to play a role in
neurodegenerative diseases such as Alzheimer's and Parkinson's. Knowledge of factors that control the rates of enzyme-catalyzed reactions thus is essential to an understanding of the molecular basis of disease. This chapter outlines the patterns by which metabolic processes are controlled and provides illustrative
examples. Subsequent chapters provide additional examples.