Richard N. Armstrong

ENZYME-CATALYZED DETOXICATION REACTIONS: MECHANISMS AND STEREOCHEMISTRY

Enzyme catalyzed detoxication reactions are one of the primary defenses organisms have against chemical insult. This article reviews current chemical approaches to understanding the cooperative role of enzymes in the metabolism of foreign compounds. Emphasis is placed on chemical and stereochemical studies which help elucidate the mechanism of action and active-site topologies of the detoxication enzymes. The stereoselectivity of the cytochromes P-450 and flavin containing monooxygenases as well as the role of hemoglobin and lipid peroxidation in the primary metabolism of xenobiotics is discussed. Current knowledge of the mechanism and stereoselectivity of epoxide hydrolase is also presented. Three enzymes involved in secondary metabolism of xenobiotics, UDP-glucuronosyltransferase, sulfotransferase and glutathione S-transferase are discussed with particular emphasis on active site topology and cooperative participation with the enzymes of primary metabolism.

Stereoselectivity of rat liver cytochrome P-450c on formation of benzo[a]pyrene 4,5-oxide

Abstract The facial selectivity of purified rat liver cytochrome P-450c toward epoxidation of benzo[a]pyrene (BP) at its 4,5-position (K-region) was determined by formation, separation and quantitation of diastereomeric trans-addition products of glutathione with enzymatically produced benzo[a]pyrene 4,5-oxide (BP 4,5-oxide). Non-enzymatic trans addition of glutathione to synthetic (+)-(4S,5R)- and (−)-(4R,5S)-BP 4,5-oxides occurs equally at carbons 4 and 5 to give two diastereomeric pairs of positional isomers. The position of the glutathionyl moiety in each isomer was determined by acetylation, oxidation of the thioether to the sulfoxide, cis -elimination of the sulfoxide, and identification of the acetoxybenzo[a]pyrene formed. Correlation of the glutathione conjugates obtained from BP 4,5-oxide derived from cytochrome P-450c catalyzed oxidation of BP with those obtained from the synthetic enantiomers indicated that ≥97% of the enzymatically formed arene oxide was the (+)-(4S,5R) enantiomer.

Investigation of the kinetic and stereochemical recognition of arene and azaarene oxides by isozymes A2 and C2 of glutathione S-transferase

The stereoselectivity of the closely related isozymes A2 and C2 of rat liver glutathione S-transferase toward several arene and azaarene oxides is examined. Isozyme C2 is stereospecific, catalyzing attack of glutathione at the oxirane carbon of R absolute configuration for a series of K-region arene oxides including phenanthrene 9,10-oxide, 1. Substitution of nitrogen in the biphenyl system of 1 causes a loss in stereospecificity. Isozyme A2 exhibits a low degree of stereoselectivity toward both arene and azaarene oxides. Kinetic studies of the two isozymes show that although isozyme C2 turns over 1 faster than does isozyme A2 the opposite is true when 4,5- diazaphenanthrene 9,10-oxide is the substrate. The kinetic and stereochemical behavior of the homodimeric isozymes A2 and C2 can be used to predict the stereoselectivity of the heterodimeric isozyme AC perhaps suggesting that catalysis is insensitive to different subunit-subunit interactions in the three isozymes.

Stereoselective formation of benz[a]anthracene (+)-(5S,6R)-oxide and (+)-(8R,9S)-oxide by a highly purified and reconstituted system containing cytochrome P-450c

Abstract The principal oxidative metabolites formed from benz[a]anthracene (BA) by the rat liver microsomal monooxygenase system are the 5,6- and 8,9-arene oxides. In order to determine the enantiomeric composition and absolute configuration of these metabolically formed arene oxides, an HPLC procedure has been developed to separate the six isomeric glutathione conjugates obtained synthetically from the individual enantiomeric arene oxides. Both (+)- and (−)-BA 5,6-oxide gave the two possible positional isomers, but only one positional isomer was formed in each case from (+)- and (−)-BA 8,9-oxide. When [ 14 C]-BA was incubated with a highly purified and reconstituted monooxygenase system containing cytochrome P-450c, and glutathione was allowed to react with the arene oxides formed, radio-active adducts were formed predominantly (>97%) from the (+)-(5S,6R) and (+)-(8R,9S) enantiomers. The present results are in accord with theoretical predictions of the steric requirements of the catalytic binding site of cytochrome P-450c.

Crystallization and a preliminary X-ray diffraction study of isozyme 3-3 of glutathione S-transferase from rat liver.

Crystals of the homodimeric isozyme 3-3 of glutathione S-transferase from rat liver have been obtained with the hanging drop method of vapor diffusion from ammonium sulfate solutions. The successful crystallization of the enzyme required the presence of both the enzyme inhibitor (9R, 10R)-9, 10-dihydro-9-(S-glutathionyl)-10-hydroxyphenanthrene and the detergent beta-octylglucopyranoside. The crystals belong to the monoclinic space group C2, with cell dimensions of a = 88.24(8) A, b = 69.44(4) A, c = 81.28(5) A, beta = 106.01(6) degrees, and contain four dimeric enzyme molecules per unit cell. The crystals diffract to at least 2.2 A and are suitable for X-ray crystallographic structure determination at high resolution.

Synthesis and characterization of the oxygen and desthio analogues of glutathione as dead-end inhibitors of glutathione S-transferase

The oxygen analogue, gamma-L-Glu-L-SerGly (GOH) and desthio analogue, gamma-L-Glu-L-AlaGly (GH) have been synthesized by a simple three step procedure involving active ester coupling of N-t-BOC-alpha-(4-nitrophenyl)-L-glutamate to L-SerGly and L-AlaGly, respectively. The two peptides are excellent dead-end inhibitors of isozymes 3-3 and 4-4 of rat liver glutathione S-transferase. At low fixed concentrations of 1-chloro-2,4-dinitrobenzene (CDNB) GOH and GH are linear competitive inhibitors of isozyme 3-3 vs glutathione with KI values of 13.0 and 116 microM, respectively. Both peptides are non-competitive (mixed-type) inhibitors vs CDNB when glutathione is the fixed substrate. Similar results are obtained with both peptides and isozyme 4-4. The results rule out ordered or ping-pong kinetic mechanisms where the electrophile adds first.

Stereoselective product inhibition of glutathione S-transferase

Isozymes 3-3 and 4-4 of rat liver glutathione S-transferase are stereoselectively inhibited by the diastereomers of 9,10-dihydro-9-glutathionyl-10-hydroxyphenanthrene, 1. The conformation of the biphenyl moiety is the same in the enzyme -1 complex as in aqueous solution with the glutathionyl and hydroxy groups in the axial positions. Isozyme 4-4 is also inhibited by the four diastereomers of 1,2-diphenyl-1-(S-glutathionyl)-2-hydroxyethane. The stereoselectivity of inhibition is modest in all cases and is manifest in both the type of inhibition as well as the magnitude of Ki.

Chapter 32. Mechanistic Aspects of Xenobiotic Metabolism as Related to Drug Design

Publisher Summary Metabolic transformations of drugs and xenobiotics are catalyzed largely, though certainly not exclusively, by a group of detoxication enzymes. Examples of such enzymes include the cytochromes P-450 and lavin, containing monooxygenases, reductases and dehydrogenases, hydrolases, including esterases and epoxide hydrolase, and a variety of transferases, such as glutathione S-transferase, UDP-glucuronyltransferase, and sulfotransferases. The participation of enzymes other than detoxication enzymes in drug metabolism is also common, because many therapeutic agents are structural mimics of endogenous compounds and are, therefore, substrates for specific enzymes normally associated with “natural” substances. Species to species differences in drug metabolism and xenobiotic metabolizing enzymes are well documented. Genetic polymorphism of, for example, human detoxication enzymes can give rise to tremendous differences in metabolism. Kinetic analysis is the single most useful tool for establishing the catalytic behavior of detoxication enzymes. For multiple substrate reactions, the order of addition of substrates and release of products can give a vague notion of the mutual orientation of the substrates or products in the active site. Conventional initial velocity steady-state kinetic measurements, along with substrate, product, and dead-end inhibition can be used to determine the kinetic mechanism. The study of oxidative transformations dominates the field of drug and xenobiotic metabolism. The central role and versatile catalytic nature of the cytochromes P-450 continues to attract enormous interest. Interesting and informative observations on the stereochemistry of cytochrome P-450-catalyzed oxidations continue to be made with enzymes from species other than man. Knowledge of metabolism has contributed significantly to the discovery of new drugs, to the design of drugs with altered metabolic and pharmacokinetic profiles, and particularly to the development of prodrugs. This knowledge includes active metabolic studies and alteration–metabolic profiles. The formation of an active species from a precursor may involve a number of enzymic transformations. Though such conversion is not particularly efficient and extrapolation of results to humans is difficult, it may prove to be an informative example of the complexities that can be encountered in the metabolic activation of a prodrug.