Aspartate Transcarbamylase: STEREOSPECIFIC RESTRICTIONS ON THE BINDING SITE FOR l-ASPARTATE

Abstract

Abstract Erythro-β-hydroxy-l-aspartate is a good substrate for the catalytic subunit of the aspartate transcarbamylase of Escherichia coli, with Km = 24 mm (approximately the same as the Km for l-aspartate) and Vmax = 0.18 mole per min per g (22% of the Vmax for l-aspartate) at pH 8 and 28°. Aminomalonate is also carbamylated by the enzyme (Km approximately 770 mm and Vmax = 0.014 mole per min per g under the same conditions), but threo-β-hydroxy-dl-aspartate, erythro, threo-β-methyl-dl-aspartate, α-methyl-dl-aspartate, l-glutamate, and O-phospho-dl-serine are all inactive, even at high concentrations of enzyme. dl-α-Methylsuccinate, d- and l-tartrate, d-aspartate, the α- and β-methylaspartates, and threo-β-hydroxy-dl-aspartate are all poor inhibitors (Ki greater than 100 mm), whereas mesotartrate (Ki = 2.7 mm at pH 7 and 28°) and d- and l-malate are good competitive inhibitors for l-aspartate. Since maleate is a much better inhibitor than fumarate, we concluded previously that l-aspartate is bound to the aspartate transcarbamylase-carbamyl phosphate complex with its carboxylates oriented approximately cis to one another. Using erythro-β-hydroxy-l-aspartate as an example, four loci can be designated on the enzyme-carbamyl phosphate complex, corresponding to the positions of the α-amino group (A), the β-hydroxyl group (B), the β-hydrogen (C), and the α-hydrogen (D). When the carboxylates are cis, A is cis to B and C is cis to D. A single set of restrictions on these four loci can be used to rationalize the specificity of the enzyme for analogues of l-aspartate as substrates or inhibitors and the previous observation that l-aspartate binds very poorly to the enzyme-carbamyl phosphate complex. The restrictions are (a) that Loci A and B can accept hydroxyl groups without substantial impairment of binding but Loci C and D cannot, and (b) that an amino or methyl group in any of the four loci undergoes an interaction with the enzyme-carbamyl phosphate complex that prevents tight binding. These restrictions lead to the conclusions that mesotartrate binds with the hydroxyl groups in Loci A and B but not in C and D, that l-malate binds with the hydroxyl group in Locus A, and that d-malate binds with the hydroxyl group in Locus B.