Identification of catalytic cysteine, histidine, and lysine residues in Escherichia coli homoserine transsuccinylase.
Homoserine transsuccinylase catalyzes the succinylation of homoserine in several bacterial species, the first unique step in methionine biosynthesis in these organisms. The enzyme from Escherichia coli is reported to be a dimer and uses a ping-pong catalytic mechanism involving transfer of succinate from succinyl-CoA to an enzyme nucleophile, followed by transfer to homoserine to form O-succinylhomoserine. Site-directed mutagenesis and steady-state kinetics were used to identify three amino acids that participate in catalysis. Mutation of cysteine-142 to serine or alanine eliminated all measurable activity, suggesting this amino acid acts as the catalytic nucleophile. Cysteine nucleophiles are often deprotonated by histidine residues, and histidine-235 was identified as the sole absolutely conserved histidine residue among family members. This residue was mutated to both alanine and asparagine, and no activity was observed with either mutant. Lysine-47 had been previously identified as an essential residue. Mutation of this amino acid to arginine reduced catalytic activity by greater than 90%, while mutation to alanine yielded an enzyme with <1% of wild-type activity. A pH-rate profile of the K47R mutant demonstrated that this amino acid participates in the first half reaction. The data presented here provide the first detailed description of the homoserine transsuccinylase active site and provide a framework for additional mechanistic characterization of this enzyme.
Ziegler
Katharine
K
Department of Chemistry & Biochemistry, George Mason University, 10900 University Boulevard, Manassas, Virginia 20110, USA.
Noble
Schroeder M
SM
Mutumanje
Elissa
E
Bishop
Barney
B
Huddler
Donald P
DP
Born
Timothy L
TL
D002384:Catalysis; D003545:Cysteine / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D006639:Histidine / Q000378:metabolism*; D051082:Homoserine O-Succinyltransferase / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007460:Iodoacetamide / Q000494:pharmacology; D008239:Lysine / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed
D006639:Histidine; D051082:Homoserine O-Succinyltransferase; D008239:Lysine; D003545:Cysteine; D007460:Iodoacetamide