Kinetic mechanism of the beta-lactam synthetase of Streptomyces clavuligerus.
Streptomyces clavuligerus beta-lactam synthetase (beta-LS) was recently demonstrated to catalyze an early step in clavulanic acid biosynthesis, the ATP/Mg(2+)-dependent intramolecular closure of the beta-amino acid N(2)-(carboxyethyl)-L-arginine (CEA) to the monocyclic beta-lactam deoxyguanidinoproclavaminic acid (DGPC). Here we investigate the steady-state kinetic mechanism of the beta-LS-catalyzed reaction to better understand this unprecedented secondary metabolic enzyme. Initial velocity patterns were consistent with a sequential ordered bi-ter kinetic mechanism. Product inhibition studies with PP(i) and DGPC demonstrated competitive inhibition versus their cognate substrates ATP and CEA, respectively, and noncompetitive inhibition against their noncognate substrates. To clarify the order of substrate binding, the truncated substrate analogue N(2)-(carboxymethyl)-L-arginine was synthesized and demonstrated uncompetitive inhibition versus ATP and competitive patterns versus CEA. These data are consistent with ordered substrate binding, with ATP binding first, an abortive enzyme-DGPC complex, and PP(i) released as the last product. The pH dependence of V and V/K was determined and suggests that residues with a pK of 6.5 and 9.3 must be ionized for optimal activity. These observations were considered in the context of investigations of the homologous primary metabolic enzyme asparagine synthetase B, and a chemical mechanism is proposed that is consistent with the kinetic mechanism.
Bachmann
B O
BO
Department of Chemistry, The Johns Hopkins University, Baltimore, Maryland 21218, USA.
Townsend
C A
CA
D000255:Adenosine Triphosphate / Q000737:chemistry; D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D001120:Arginine / Q000031:analogs & derivatives* / Q000737:chemistry; D001667:Binding, Competitive; D002384:Catalysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity
C408696:N(omega)-carboxymethylarginine; C005394:octopine; D000255:Adenosine Triphosphate; D001120:Arginine; D000581:Amidohydrolases; C114020:beta-lactam synthetase