title: Partial randomization of the four sequential amidation reactions catalyzed by cobyric acid synthetase with a single point mutation.
abstract: Cobyric acid synthetase (CbiP) from Salmonella typhimurium catalyzes the glutamine and ATP-dependent amidation of carboxylates b, d, e, and g within adenosyl cobyrinic acid a,c-diamide. After each round of catalysis the partially amidated intermediates are released into solution and the four carboxylates are amidated in the sequential order of e, d, b, and g for the wild type enzyme. In the presence of [gamma-18O4]-ATP and adenosyl cobyrinic a,c-diamide the enzyme will catalyze the positional isotope exchange of the betagamma-bridge oxygen with the two beta-nonbridge oxygens. These results support the proposal that ATP is used to activate the carboxylate groups via the formation of a phosphorylated intermediate. CbiP catalyzes the hydrolysis of glutamine in the absence of ATP or adenosyl cobyrinic acid a,c-diamide, but the rate of glutamine hydrolysis is enhanced by a factor of 60 in the presence of these two substrates together. This result suggests that the formation of the phosphorylated intermediate is coupled to the activation of the site utilized for the hydrolysis of glutamine. However, the rate of glutamine hydrolysis is approximately 2.5 times the rate of ADP formation, indicating that the two active sites are partially uncoupled from one another and that some of the ammonia from glutamine hydrolysis leaks into the bulk solution. The mutation of D146 to either alanine or asparagine results in a protein that is able to catalyze the formation of cobyric acid. However, the strict amidation order observed with the wild type CbiP is partially randomized with carboxylate b being amidated last. With the D146N mutant, the predominant pathway occurs in the sequence d, e, g, and b. It is proposed that this residue enforces the amidation order in the wild type enzyme via charge-charge repulsion between the side chain carboxylate and the carboxylates of the substrate.
authors.affiliation: Department of Chemistry, P.O. Box 30012, Texas A&M University, College Station, Texas 77845-3012, USA.
authors.forename: Vicente
authors.initials: V
authors.lastname: Fresquet
authors.forename: LaKenya
authors.initials: L
authors.lastname: Williams
authors.forename: Frank M
authors.initials: FM
authors.lastname: Raushel
mesh_terms: D003038:Cobamides / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D017354:Point Mutation; D012486:Salmonella typhimurium / Q000201:enzymology; D000637:Transaminases / Q000235:genetics* / Q000378:metabolism*
chemical_list: D003038:Cobamides; C526457:adenosyl cobyrinic acid a,c-diamide; D000637:Transaminases; C071151:cobyric acid synthase