PYRIDOXAL PHOSPHATE ENZYMES: Mechanistic, exercises/5GOT/PLP enzyme... · Mechanistic, Structural,

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PYRIDOXAL PHOSPHATE ENZYMES:Mechanistic, Structural, and EvolutionaryConsiderations

Andrew C. Eliot1 and Jack F. Kirsch21Department of Chemistry and 2Departments of Chemistry and Molecular and CellBiology, University of California, Berkeley, California 94720-3206; email:aeliot@life.uiuc.edu, jfkirsch@uclink.berkeley.edu

Key Words substrate specificity, reaction type specificity, enzyme inhibition,enzyme mechanism

f Abstract Pyridoxal phosphate (PLP)-dependent enzymes are unrivaled in thediversity of reactions that they catalyze. New structural data have paved the way fortargeted mutagenesis and mechanistic studies and have provided a framework forinterpretation of those results. Together, these complementary approaches yield newinsight into function, particularly in understanding the origins of substrate andreaction type specificity. The combination of new sequences and structures enablesbetter reconstruction of their evolutionary heritage and illuminates unrecognizedsimilarities within this diverse group of enzymes. The important metabolic roles ofmany PLP-dependent enzymes drive efforts to design specific inhibitors, which arenow guided by the availability of comprehensive structural and functional databases.Better understanding of the function of this important group of enzymes is crucial notonly for inhibitor design, but also for the design of improved protein-based catalysts.

CONTENTS

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384MECHANISTIC VERSATILITY OF PLP . . . . . . . . . . . . . . . . . . . . . . . . . 385STRUCTURAL DIVERSITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387DETERMINANTS OF REACTION TYPE . . . . . . . . . . . . . . . . . . . . . . . . . 394

Dunathan Stereoelectronic Hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . 394Transamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395Racemization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 398Role of the Closed Conformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399

DETERMINANTS OF SUBSTRATE SPECIFICITY. . . . . . . . . . . . . . . . . . . 399Role of the Closed Conformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399Dual Specificity of Aminotransferases . . . . . . . . . . . . . . . . . . . . . . . . . . 399

EVOLUTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403MECHANISMS OF INHIBITION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 406

Annu. Rev. Biochem. 2004. 73:383415doi: 10.1146/annurev.biochem.73.011303.074021

Copyright 2004 by Annual Reviews. All rights reservedFirst published online as a Review in Advance on April 2, 2004

3830066-4154/04/0707-0383$14.00

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Noncovalent Inactivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 406Activated Nucleophiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 408Activated Electrophiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409Reversible Competitive Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410The Challenge of Inhibitor Specificity . . . . . . . . . . . . . . . . . . . . . . . . . . 411

CONCLUSIONS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411

INTRODUCTION

Following its identification in 1951 as one of the active vitamers of vitamin B6(1), pyridoxal 5-phosphate (PLP) has been the subject of extensive researchdirected toward understanding its unequaled catalytic versatility.1 As a result, thebasic mechanisms of PLP-assisted reactions, both in solution and enzyme-associated, have been well characterized and are now a staple of biochemistrytextbooks (for example, see 2, 3). A number of reviews have addressed thesubject in greater detail, and readers are directed particularly to the recent articlesby Hayashi (4) and John (5). Transaminases, edited by Christen & Metzler (6),remains an excellent and thorough source of information about these enzymes.More detailed reviews concentrate on the function of a number of specificenzymes, including tryptophan synthase (7), O-acetylserine sulfhydrylase (8),-aminolevulinate synthase (9), serine hydroxymethyltransferase (SHMT) (10),and branched chain amino acid aminotransferase (BCAT) (11).

In addition to their versatility as catalysts, PLP-dependent enzymes haveattracted attention because of their widespread involvement in cellular processes.These enzymes are principally involved in the biosynthesis of amino acids andamino acid-derived metabolites, but they are also found in the biosyntheticpathways of amino sugars (12) and other amine-containing compounds. Theirimportance is further underscored by the number identified as drug targets. Forexample, inhibitors of -aminobutyric acid aminotransferase (GABA ATase) areused in the treatment of epilepsy (13), SHMT has been identified as a target forcancer therapy (14), and inhibitors of ornithine decarboxylase (ODC) areemployed in the treatment of African sleeping sickness (15). Functional defectsin PLP enzymes have furthermore been implicated in a number of diseasepathologies, including homocystinuria, which is most frequently caused bymutations in cystathionine -synthase (16, 17).

1Abbreviations: AATase, aspartate aminotransferase; ACC, 1-aminocyclopropane-1-carboxylate; AlaP, 1-aminoethylphosphonate; ALR, alanine racemase; AONS, 8-amino-7-oxononanoate synthase; ATase, aminotransferase; BCAT, branched chain amino acidaminotransferase; DAAT, D-amino acid aminotransferase; DGD, dialkylglycine decar-boxylase; GABA, -aminobutyric acid; OAT, ornithine aminotransferase; ODC, ornithinedecarboxylase; PLP, pyridoxal 5-phosphate; PMP, pyridoxamine 5-phosphate; SAM,S-adenosyl-L-methionine; SHMT, serine hydroxymethyl transfease; and TATase, aro-matic amino acid aminotransferase.

384 ELIOT y KIRSCH

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Despite the long history of research in the field, we are only now beginningto answer some of the most exciting questions. In particular, technologicaladvances that have allowed ever more rapid determination of enzyme structuresand the accumulation of large sequence databases have also enhanced ourunderstanding of enzymatic catalysis. Analyses of sequences and structures yieldsignificant insight regarding the evolution of this diverse class of enzymes.Additionally, crystal structures elegantly demonstrate how PLP enzymes harnessthe potential of the cofactor to accelerate the rates only of specific reactions, andthey provide the basis for targeted mutagenesis.

This review describes many of the new insights that have come from recentstructural and mechanistic studies, with a primary focus on determinants ofsubstrate specificity and reaction type, as well as the design of inhibitors for thisclass of enzyme. The mechanisms and structures are briefly discussed to providebackground.

MECHANISTIC VERSATILITY OF PLP

PLP-catalyzed reaction types can be divided according to the position at whichthe net reaction occurs. Reactions at the position include transamination,decarboxylation, racemization, and elimination and replacement of an electro-philic R group. Those at the or position include elimination or replacement.Examples of each of these reactions are shown in Scheme 1, and the basicmechanisms are shown in Schemes 2 () and 3 ( and ). Exceptions to thesecommon types include the formation of a cyclopropane ring from S-adenosyl-L-methionine (SAM), catalyzed by 1-aminocyclopropane-1-carboxylate (ACC)synthase (18), and the cleavage of ACC to -ketobutyrate and ammonia,catalyzed by ACC deaminase (19); these are not discussed here. Because manyof the reaction pathways share common intermediates, a number of enzymes alsocatalyze reactions that are combinations of the basic types, such as the decar-boxylation-dependent transamination (aminoisobutyrate pyruvate 3 acetone CO2 L-Ala) catalyzed by dialkylglycine decarboxylase (DGD; 20) or the-elimination and -replacement (O-phospho-L-homoserine H2O 3 L-Thr Pi) catalyzed by threonine synthase (21).

Although the scope of PLP-catalyzed reactions initially appears to be bewil-deringly diverse, there is a simple unifying principle. The cofactor in all casesfunctions to stabilize negative charge development at C in the transition statethat is formed after condensation of the amino acid substrate with PLP to forma Schiff base (referred to as the external aldimine).2 The fully formed carbanion

2There are two exceptions to this basic principle. The glycogen phosphorylase family ofenzymes utilizes the phosphate group of PLP for catalysis [reviewed in (106)], and theaminomutase family catalyzes a radical-initiated reaction on PLP-bound am