Beta-lactamase-catalyzed aminolysis of depsipeptides: Proof of the nonexistence of a specific D-phenylalanine/enzyme complex by double-label isotope trapping

Journal Article · Tue Aug 22 04:00:00 EDT 1989 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00443a014· OSTI ID:5075034

Pazhanisamy, S; Pratt, R F ^[1]

---

1. Wesleyan Univ., Middletown, CT (USA)

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. 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.

• OSTI ID: 5075034
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 28:17; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMIDASES
AMINO ACIDS
BACTERIA
BIOCHEMICAL REACTION KINETICS
CARBOXYLIC ACIDS
DICHROISM
DOUBLE LABELLING
ENZYME ACTIVITY
ENZYMES
FLUORESCENCE
GLYCINE
HYDROGEN COMPOUNDS
HYDROLASES
ISOTOPE APPLICATIONS
ISOTOPE DILUTION
KINETICS
LABELLING
LUMINESCENCE
MAGNETIC CIRCULAR DICHROISM
MAGNETIC RESONANCE
METABOLISM
MICROORGANISMS
NON-PEPTIDE C-N HYDROLASES
NUCLEAR MAGNETIC RESONANCE
ORGANIC ACIDS
ORGANIC COMPOUNDS
PEPTIDES
PHENYLALANINE
PROTEINS
REACTION KINETICS
RESONANCE
STEADY-STATE CONDITIONS
TRACER TECHNIQUES
TRAPPING
TRITIUM COMPOUNDS