Reactions of Alternate Substrates Demonstrate Stereoelectronic Control of Reactivity in Dialkylglycine Decarboxylase Shaoxian Sun, Roger F. Zabinski, and Michael D. Toney* Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park AVenue, Bronx, New York 10461 ReceiVed August 19, 1997; ReVised Manuscript ReceiVed December 18, 1997 ABSTRACT: Kinetic and product analyses of the reactions of dialkylglycine decarboxylase with several alternative substrates are presented. Rate constants for the reactions of amino and keto acids of several substrates decrease logarithmically with increasing side-chain size. Conversely, k cat for L-amino acid decarboxylation increases with side-chain size. These and other data confirm a proposed model for three binding subsites in the active site. In this model, bond making and breaking in both the decarboxylation and transamination half-reactions occurs at the “A” subsite, which maintains the scissile bond aligned with the p orbitals of the conjugated aldimine and thus maximizes stereoelectronic effects. This strongly supports the proposal by Dunathan (Proc. Natl. Acad. Sci. U.S.A. 55, 712-716) that PLP-dependent enzymes can largely control reaction specificity by specific orientation about C R in the external aldimine intermediate. The “B” subsite can accept either an alkyl or a carboxylate group, while the “C” subsite accepts only small alkyl groups. This model predicts the existence of nonproductive binding modes for amino acids, which is proposed to be the ultimate origin of the k cat increase with side-chain size for L-amino acid decarboxylation. The specificity of the 2-aminoisobutyrate decarboxylation half-reaction toward oxidative decarboxylation is very high (<1 in 10 5 turnovers yields nonoxidative decarboxylation). The origin of this specificity is explored with the reactions of amino- and methylaminomalonate. These substrates exhibit high yields of nonoxidative decarboxylation, providing support for a model in which the interaction between a carboxylate group in the B subsite and Arg406 is a prerequisite to proton donation to and removal from C R . Dialkylglycine decarboxylase (DGD) 1,2 from Pseudomonas cepacia is a PLP dependent enzyme that catalyzes the oxidative decarboxylation of 2,2-dialkylglycines, yielding CO 2 , a ketone, and the PMP form of the enzyme (Scheme 1). The catalytic cycle is completed by transamination of an R-keto acid, preferentially pyruvate (1-3). The overall reaction catalyzed by DGD is shown below. The unusual ability of DGD to catalyze rapidly two classical PLP- dependent reactions has rekindled an interest in this enzyme. The X-ray structure of DGD has been determined to 2.1-Å resolution (4, 5). The fold of the enzyme is roughly similar to the well-studied aspartate aminotransferase (6), which is a member of evolutionary subgroup I of aminotransferases (7). DGD is a representative member of evolutionary subgroup II (8), and its fold is closely similar to the other known structures of subgroup II enzymes (9, 10). The active site structure of DGD is similar to that of aspartate ami- notransferase in that the interactions made between coenzyme and protein are highly conserved (5). Toney et al. (5) proposed a structurally based model of the active site of DGD in which there are three binding subsites (A-C) that differ in their functional group specifici- ties. One of these subsites, the A subsite, was proposed to be the locus of all bond-making/-breaking events. This model makes predictions about the reaction specificity of various substrate analogues and largely provided the impetus for the present study. PLP-dependent enzymes catalyze a wide variety of reac- tions at the R-, -, and γ-carbons of amino acids and amines. Dunathan (11) proposed that reaction specificity in PLP enzymes is largely controlled by specific orientation about the C R -N bond in the external aldimine intermediate. Stereoelectronic effects, and thereby reaction rates, are maximized when a bond to C R is perpendicular to the plane of the PLP ring, i.e., when the bond has maximal overlap with the conjugated π system of the aldimine. Direct experimental evidence for this proposal has been sparse. It includes the observations that the C R -CH 3 group of the competitive inhibitor 2-methylaspartate in the active site of aspartate aminotransferase is so oriented (12), that arginine * Corresponding author: (e-mail) toney@aecom.yu.edu; (tel) 718- 430-2347; (fax) 718-430-8565. 1 M.D.T. dedicates this paper to Professor J. F. Kirsch for his patience and wisdom as a teacher. Supported by Grant NIGMS 54779. 2 Abbreviations: DGD, dialkylglycine decarboxylase; PLP, pyridoxal phosphate; PMP, pyridoxamine phosphate; DGD-PLP, PLP form of DGD; DGD-PMP, PMP form of DGD; AIB, R-aminoisobutyrate; AC3C, 1-amino-1-cyclopropanecarboxylate; AC5C, 1-amino-1-cyclo- pentane-carboxylate; AC6C, 1-amino-1-cyclohexanecarboxylate; 2°ADH, secondary alcohol dehydrogenase; YADH, yeast alcohol dehydrogenase; LDH, lactate dehydrogenase; LADH, liver alcohol dehydrogenase; MAM, R-methyl-R-aminomalonate; AM, R-aminomalonate; TEA-HCl, triethanolamine hydrochloride. 3865 Biochemistry 1998, 37, 3865-3875 S0006-2960(97)02055-2 CCC: $15.00 © 1998 American Chemical Society Published on Web 02/26/1998