Beta-secondary and solvent deuterium kinetic isotope effects on beta-lactamase catalysis.
Beta-Secondary and solvent deuterium kinetic isotope effects have been determined for the steady-state kinetic parameters V/K and V for turnover of a depsipeptide substrate, m-[[(phenylacetyl)glycyl]-oxy]benzoic acid, and of a beta-lactam substrate, penicillanic acid, by three typical class A beta-lactamases and a class C beta-lactamase. The isotope effects on alkaline hydrolysis of these substrates have been used as a frame of reference. The effect of the transition state conformation of the substrates in determining the beta-secondary isotope effects has been explicitly considered. The inverse beta-secondary isotope effects on both V/K and V for the class A enzymes with both substrates indicate transition states where the carbonyl group of the scissile bond has become tetrahedral and therefore reflect typical acyl-transfer transition states. The solvent isotope effects indicate that enzyme deacylation (as reflected in V for the Staphylococcus aureus PC1 beta-lactamase) may be a classical general-base-catalyzed hydrolysis but that there is little proton motion in the enzyme acylation transition state (as revealed by V/K) for the TEM beta-lactamase and Bacillus cereus beta-lactamase I. These results provide kinetic support for the conjecture made on structural grounds that class A beta-lactamases employ an asymmetric double-displacement mechanism. The isotope effects on V/K for the class C beta-lactamase of Enterobacter cloacae P99 suggest an acyl-transfer transition state for the penicillin, although, as for the class A enzymes, without significant proton motion. On the other hand, the V/K transition state for depsipeptide does not seem to involve covalent chemistry. Suggestive of this conclusion are the measured beta-secondary isotope effect of 1,002 +/- 0.012 and the inverse solvent isotope effect. These results provide an example of a significant difference between the kinetics of turnover of a beta-lactam and a depsipeptide by a beta-lactamase. The V transition state for both substrates with the P99 beta-lactamase probably involves acyl-transfer (deacylation) where the conformation of the acyl-enzyme is closely restricted. The conformations of acyl-enzymes of the PC1 and P99 beta-lactamases correlate to the (different) dispositions of general base catalysts at their active sites.
Adediran
S A
SA
Department of Chemistry, Wesleyan University, Middletown, Connecticut 06459, USA.
Deraniyagala
S A
SA
Xu
Y
Y
Pratt
R F
RF
D000215:Acylation; D001409:Bacillus cereus / Q000201:enzymology; D003903:Deuterium; D004754:Enterobacter / Q000201:enzymology; D006868:Hydrolysis; D007700:Kinetics; D009038:Motion; D010455:Peptides / Q000378:metabolism; D017433:Protein Structure, Secondary; D011522:Protons; D012997:Solvents; D013211:Staphylococcus aureus / Q000201:enzymology; D001618:beta-Lactamases / Q000737:chemistry* / Q000378:metabolism
D010455:Peptides; D011522:Protons; D012997:Solvents; D003903:Deuterium; D001618:beta-Lactamases