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 00:00:00 EDT 1989 · Biochemistry; (USA)

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

Pazhanisamy, S; Pratt, R F ^[1]

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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), Vol. 28:17; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

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