IDENTIFICATION OF CRITICAL AMINO-ACID-RESIDUES OF SACCHAROMYCES-CEREVISIAE CARBAMOYL-PHOSPHATE SYNTHETASE - DEFINITION OF THE ATP SITE INVOLVED IN CARBOXY-PHOSPHATE FORMATION

Carbamoyl-phosphate synthetases (CPSases) utilize two molecules of ATP at two homologous domains, B and C, with ATP, used to form the enzyme -bound intermediate carboxy-phosphate and ATP(C) used to phosphorylate the carbamate intermediate. To further define the role of one CPSase peptide suggested by affinity labeling studies to be near the ATP(B) s ite, we have carried out site-directed mutagenic analysis of peptide 2 34-242 of the Saccharomyces cerevisiae arginine-specific CPSase. Mutan ts E234A, E234D, E236A, E236D and E238A were unable to complement the CPSase-deficient yeast strain LPL26 whereas mutants Y237A, E238D, R241 K, R241E and R241P supported LPL26 growth as well as wild-type CPSase. Kinetic analysis of E234A and Y237A indicated impaired utilization of ATP(B) but not of ATP(C). D242A, a temperature-sensitive mutant, reta ined no detectable activity when assayed in vitro. These findings, tog ether with the affinity labeling data and primary sequence analysis, s trongly suggest that the yeast CPSase peptide 234-242 is located at th e ATP(B) site and that some of its residues are important for function ing of the enzyme. D242 appears to occupy a critical structural positi on and E234, E236 and E238 appear to be critical for function, with th e spatial arrangement of the carboxyl side chain also critical for E23 4 and E236. (C) 1997 Elsevier Science B.V.