Kinetics and inhibition of glutamate carboxypeptidase II using a microplate assay

Camilo Rojas, Scott T. Frazier, Juliet Flanary, Barbara S. Slusher

Research output: Contribution to journalArticle

Abstract

Glutamate carboxypeptidase II (GCPII or prostate-specific membrane antigen or NAALADase) is an enzyme that catalyzes the hydrolysis of the neuropeptide N-acetylaspartylglutamate (NAAG) to N-acetylaspartate (NAA) and glutamate (G). Inhibitors of GCPII provide neuroprotection in a variety of animal models of central nervous system disorders. Neuroprotection is probably the result of increased NAAG concentrations and decreased levels of excess toxic glutamate. Consequently, GCPII inhibitors could be useful therapeutic agents where increased glutamate levels are the result of increased GCPII activity. Current GCPII in vitro activity assays are cumbersome or have limited sensitivity. In this report we describe a microplate assay to study GCPII inhibition that is most sensitive, efficient, and generates little waste. GCPII turnover number (kcat) was 4 s-1 and the binding constant (Km) for NAAG and GCPII was 130 nM. The apparent association rate constant for GCPII and NAAG (kcat/Km) was 3 x 107 M-1 s-1. Inhibition studies with the GCPII inhibitor 2-(phosphonomethyl)pentanedioic acid (2-PMPA) demonstrated competitive inhibition with a Ki = 0.2 nM.

Original languageEnglish (US)
Pages (from-to)50-54
Number of pages5
JournalAnalytical biochemistry
Volume310
Issue number1
DOIs
StatePublished - Nov 1 2002
Externally publishedYes

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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