Reaction mechanism of the fructose 2,6-bisphosphatase

H. Mizuguchi, F. F. Cook, C. A. Hasemann, K. Uyeda, V. A. Dallas

Research output: Contribution to journalArticle

Abstract

A bifunctional enzyme, Fru 6-P,2-kinase:Fru 2,6-Pase, catalyzes synthesis and degradation of Fru 2,6-Pj. The rat liver Fru 2,6-Pase reaction (Fru 2,6-P2 -Fru 6-P + P,) proceeds via a phosphoryl-His258 intermediate. Recently we found that mutation of the corresponding His256 of rat testis enzyme did not significantly affect the activity. We have prepared additional mutant enzymes and investigated the reaction mechanism in more detail. Mutant enzymes H256A and H390A showed: (a) Vm values of 17% and 5%, respectively, of the wild type enzymes; and (b) Fru 6-P and P respectively, were noncompetitive and competitive inhibitors with respect to Fru 2,6-P2, as are wild type, suggesting that all these enzymes follow the same reaction pathway via a P-enzyme intermediate. A double mutant H256A/H390A was completely inactive. The initial rate of Fru 6-P formation by H390A was biphasic with an initial burst followed by a linear rate, while that of P| formation was linear. It was possible to isolate a P-enzyme intermediate (P-His256) in stoichiometric amounts, indicating that the burst was due to the P-enzyme formation. However, no such burst was observed with H256A and the wild type enzymes. Attempts to isolate a P-His intermediate with the H256A mutant were unsuccessful. We suggest that Fru 2,6-Pase can catalyze by two alternative pathways, using either His256 or His390 as a phosphoryi-acceptor or a direct hydrolysis involving His390 as a base. (Supported by grants from the DVA, NIDDK, and NIGMS).

Original languageEnglish (US)
Pages (from-to)A1444
JournalFASEB Journal
Volume12
Issue number8
StatePublished - Dec 1 1998

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

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  • Cite this

    Mizuguchi, H., Cook, F. F., Hasemann, C. A., Uyeda, K., & Dallas, V. A. (1998). Reaction mechanism of the fructose 2,6-bisphosphatase. FASEB Journal, 12(8), A1444.