Structure of a Michaelis complex analogue: Propionate binds in the substrate carboxylate site of alanine racemase

The structure of alanine racemase from Bacillus stearothermophilus with the inhibitor propionate bound in the active site was determined by X-ray crys tallography to a resolution of 1.9 Angstrom. The enzyme is a homodimer in s olution and crystallizes with a dimer in the asymmetric unit. Both active s ites contain a pyridoxal 5'-phosphate (PLP) molecule in aldimine linkage to Lys39 as a protonated Schiff base, and the pH-independence of UV-visible a bsorption spectra suggests that the protonated PLP-Lys39 Schiff base is the reactive form of the enzyme. The carboxylate group of propionate bound in the active site makes numerous interactions with active-site residues, defi ning the substrate binding site of the enzyme. The propionate-bound structu re therefore approximates features of the Michaelis complex formed between alanine racemase and its amino acid substrate. The structure also provides evidence for the existence of a carbamate formed on the side-chain amino gr oup of Lys129, stabilized by interactions with one of the residues interact ing with the carboxylate group of propionate, Arg136. We propose that this novel interaction influences both substrate binding and catalysis by precis ely positioning Arg136 and modulating its charge.