Substrate specificity, inhibition and enzymological analysis of recombinant human glutamate carboxypeptidase II

Cyril Barinka, Markéta Rinnová, Pavel Šácha, Camilo Rojas, Pavel Majer, Barbara S. Slusher, Jan Konvalinka

Research output: Contribution to journalArticle

Abstract

Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a membrane peptidase expressed in a number of tissues such as kidney, prostate and brain. The brain form of GCPII (also known as NAALADase) cleaves N-acetyl-aspartyl glutamate to yield free glutamate. Animal model experiments show that inhibition of GCPII prevents neuronal cell death during experimental ischaemia. GCPII thus represents an important target for the treatment of neuronal damage caused by excess glutamate. In this paper we report expression of an extracellular portion of human glutamate carboxypeptidase II (amino acids 44-750) in Drosophila Schneider's cells and its purification to homogeneity. A novel assay for hydrolytic activity of recombinant human GCPII (rhGCPII), based on fluorimetric detection of released alpha-amino groups was established, and used for its enzymological characterization. rhGCPII does not show dipeptidylpeptidase IV-like activity assigned to the native form of the enzyme previously. Using a complete set of protected dipeptides, substrate specificity of rhGCPII was elucidated. In addition to the previously described substrates, four novel compounds, Ac-Glu-Met, Ac-Asp-Met and, surprisingly, Ac-Ala-Glu and Ac-Ala-Met were identified as substrates for GCPII, and their respective kinetic constants determined. The glycosylation of rhGCPII was found indispensable for the enzymatic activity.

Original languageEnglish (US)
Pages (from-to)477-487
Number of pages11
JournalJournal of Neurochemistry
Volume80
Issue number3
DOIs
StatePublished - Feb 2002
Externally publishedYes

Keywords

  • Enzyme glycosylation
  • Glutamate carboxypeptidase II
  • NAALADase
  • Neuroprotection
  • Zinc metallopeptidase

ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

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