Inhibition of TEM-2 beta-lactamase from Escherichia coli by clavulanic acid: observation of intermediates by electrospray ionization mass spectrometry

Biochemistry. 1996 Sep 24;35(38):12421-32. doi: 10.1021/bi961044g.

Abstract

Clavulanic acid, the therapeutically important inhibitor of beta-lactamases containing a nucleophilic serine residue at their active sites, inhibits Escherichia coli TEM-2 beta-lactamase via a complex mechanism. Electrospray ionization mass spectrometry (ESIMS) studies revealed that a minimum of four different modified proteins are formed upon incubation of clavulanate with the TEM-2 enzyme. These exhibit mass increments relative to the unmodified TEM-2 beta-lactamase of 52, 70, 88, and 155 Da. Time course studies implied that no long-lived forms of clavulanate-inhibited TEM-2 beta-lactamase retain the carbons of the oxazolidine ring of clavulanate. The absence of a 199 Da increment to unmodified TEM-2 suggests rapid decarboxylation of clavulanate upon binding to the enzyme. Proteolytic digestions of purified forms of clavulanate inhibited TEM-2 beta-lactamase followed by analyses using high-performance liquid chromatography coupled to ESIMS (HPLC-ESIMS) and chemical sequencing were used to provide positional information on the modifications to the enzyme. Increments of 70 and 80 Da increments were shown to be located in a peptide containing Ser-70. A further 70 Da mass increment, assigned as a beta-linked acrylate, was localized to a peptide containing Ser-130. A mechanistic scheme for the reaction of clavulanate with TEM-2 beta-lactamase is proposed in which acylation at Ser-70 and subsequent decarboxylation is followed either by cross-linking with Ser-130 to form a vinyl ether or by reformation of unmodified enzyme via a Ser-70 linked (hydrated) aldehyde. Purified cross-linked vinyl ether was observed to slowly convert under acidic conditions to a Ser-70 linked (hydrated) aldehyde with concomitant conversion of Ser-130 to a dehydroalanyl residue.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Clavulanic Acid
  • Clavulanic Acids / chemistry
  • Clavulanic Acids / metabolism
  • Clavulanic Acids / pharmacology*
  • Dithiothreitol / metabolism
  • Enzyme Inhibitors / pharmacology*
  • Escherichia coli / enzymology
  • Kinetics
  • Mass Spectrometry
  • Models, Chemical
  • Molecular Structure
  • Penicillanic Acid / analogs & derivatives
  • Penicillanic Acid / pharmacology
  • Serine / metabolism
  • Serine Endopeptidases / metabolism
  • Trypsin / metabolism
  • beta-Lactamase Inhibitors*
  • beta-Lactamases / chemistry
  • beta-Lactamases / metabolism

Substances

  • Clavulanic Acids
  • Enzyme Inhibitors
  • beta-Lactamase Inhibitors
  • Clavulanic Acid
  • Serine
  • Penicillanic Acid
  • 6-iodopenicillanic acid
  • Serine Endopeptidases
  • glutamyl endopeptidase
  • Trypsin
  • beta-Lactamases
  • beta-lactamase TEM-2
  • Dithiothreitol