Rate-limiting steps and role of active site Lys443 in the mechanism of carbapenam synthetase.
Carbapenam synthetase (hereafter named CPS) catalyzes the formation of the beta-lactam ring in the biosynthetic pathway to (5R)-carbapen-2-em-3-carboxylate, the simplest of the carbapenem antibiotics. Kinetic studies showed remarkable tolerance to substrate stereochemistry in the turnover rate but did not distinguish between chemistry and a nonchemical step such as product release or conformational change as being rate-determining. Also, X-ray structural studies and modest sequence homology to beta-lactam synthetase, an enzyme that catalyzes the formation of a monocyclic beta-lactam ring in a similar ATP/Mg2+-dependent reaction, implicate K443 as an essential residue for substrate binding and intermediate stabilization. In these experiments, we use pH-rate profiles, deuterium solvent isotope effects, and solvent viscosity measurements to examine the rate-limiting step in this complex overall process of substrate adenylation and intramolecular ring formation. Mutagenesis and chemical rescue demonstrate that K443 is the general acid visible in the pH-rate profile of the wild-type CPS-catalyzed reaction. On the basis of these results, we propose a mechanism in which the rate-limiting step is beta-lactam ring formation coupled to a protein conformational change and underscore the role of K443 throughout the reaction.
Department of Chemistry, The Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, USA.
Samantha O
SO
Arnett
Barbara
B
Gerratana
Craig A
CA
Townsend
D000409:Alanine / Q000235:genetics; D001665:Binding Sites; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D003903:Deuterium / Q000737:chemistry; D004789:Enzyme Activation / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014644:Genetic Variation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D012997:Solvents / Q000737:chemistry; D013379:Substrate Specificity / Q000235:genetics; D014783:Viscosity
D029968:Escherichia coli Proteins; D012997:Solvents; D003903:Deuterium; D019731:Carbon-Nitrogen Ligases; C479139:carbapenam synthetase; D008239:Lysine; D000409:Alanine