Beta-lactamase-catalyzed aminolysis of depsipeptides: proof of the nonexistence of a specific D-phenylalanine/enzyme complex by double-label isotope trapping.
The steady-state kinetics of the Enterobacter cloacae P99 beta-lactamase-catalyzed aminolysis of the depsipeptide m-[[(phenylacetyl)glycyl]oxy]benzoic acid by D-phenylalanine were consistent with an ordered sequential mechanism with D-phenylalanine binding first [Pazhanisamy, S., Govardhan, C. P., & Pratt, R. F. (1989) Biochemistry (first of three papers in this issue)]. In terms of this mechanism, the kinetics data required that in 20 mM MOPS buffer, pH 7.5, the dissociation constant of the initially formed enzyme/D-phenylalanine complex be around 1.3 mM; at pH 9.0 in 0.1 M carbonate buffer, the complex should be somewhat more stable. Attempts to detect this complex in a binary mixture by spectroscopic methods (fluorescence, circular dichroic, and nuclear magnetic resonance spectra) failed. Kinetic methods were also unsuccessful--the presence of 20 mM D-phenylalanine did not appear to affect beta-lactamase activity nor inhibition of the enzyme by phenylmethanesulfonyl fluoride, phenylboronic acid, or (3-dansylamidophenyl)boronic acid. Equilibrium dialysis experiments appeared to indicate that the dissociation constant of any binary enzyme/D-phenylalanine complex must be somewhat higher than the kinetics allowed (greater than 2 mM). Since the kinetics also required that, at high depsipeptide concentrations, and again with the assumption of the ordered sequential mechanism, the reaction of the enzyme/D-phenylalanine complex to aminolysis products be faster than its reversion to enzyme and D-phenylalanine, a double-label isotope-trapping experiment was performed.(ABSTRACT TRUNCATED AT 250 WORDS)
Pazhanisamy
S
S
Department of Chemistry, Wesleyan University, Middletown, Connecticut 06457.
Pratt
R F
RF
D002942:Circular Dichroism; D003956:Dialysis; D004754:Enterobacter / Q000201:enzymology*; D004755:Enterobacteriaceae / Q000201:enzymology*; D005998:Glycine / Q000378:metabolism / Q000031:analogs & derivatives; D007536:Isomerism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010648:Phenylacetates / Q000378:metabolism*; D010649:Phenylalanine; D011485:Protein Binding; D011865:Radioisotope Dilution Technique; D013050:Spectrometry, Fluorescence; D014316:Tritium; D001618:beta-Lactamases / Q000378:metabolism*
D010648:Phenylacetates; D014316:Tritium; C061747:3-(((phenylacetyl)glycyl)oxy)benzoic acid; D010649:Phenylalanine; D001618:beta-Lactamases; D005998:Glycine