Lysine 213 and histidine 233 participate in Mn(II) binding and catalysis in Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase

Hans Krautwurst, Hannetz Roschzttardtz, Sergio Bazaes, Fernando D. González-Nilo, Thomas Nowak, Emilio Cardemil

Research output: Contribution to journalArticlepeer-review

14 Citations (Scopus)

Abstract

Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase catalyses the reversible metal-dependent formation of oxaloacetate and ATP from PEP, ADP, and CO2 and plays a key role in gluconeogenesis. This enzyme also has oxaloacetate decarboxylase and pyruvate kinase-like activities. Mutations of PEP carboxykinase have been constructed where the residues Lys213 and His233, two residues of the putative Mn2+ binding site of the enzyme, were altered. Replacement of these residues by Arg and by Gln, respectively, generated enzymes with 1.9 and 2.8 kcal/mol lower Mn2+ binding affinity. Lower PEP binding affinity was inferred for the mutated enzymes from the protection effect of PEP against urea denaturation. Kinetic studies of the altered enzymes show at least a 5000-fold reduction in Vmax for the primary reaction relative to that for the wild-type enzyme. Vmax values for the oxaloacetate decarboxylase and pyruvate kinase-like activities of PEP carboxykinase were affected to a much lesser extent in the mutated enzymes. The mutated enzymes show a decreased steady-state affinity for Mn2+ and PEP. The results are consistent with Lys213 and His233 being at the Mn2+ binding site of S. cerevisiae PEP carboxykinase and the Mn2+ affecting the PEP interaction. The different effects of mutations in Vmax for the main reaction and the secondary activities suggest different rate-limiting steps for these reactions.

Original languageEnglish
Pages (from-to)12763-12770
Number of pages8
JournalBiochemistry
Volume41
Issue number42
DOIs
Publication statusPublished - 22 Oct 2002

ASJC Scopus subject areas

  • Biochemistry

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