Lysine 213 and Histidine 233 Participate in Mn(II) Binding and Catalysis in Saccharomyces cerevisiae Phosphoenolpyruvate Carboxykinase

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• 作者：Hans Krautwurst ; Hannetz Roschzttardtz ; Sergio Bazaes ; Fernando D. Gonz&aacute ; lez-Nilo ; Thomas Nowak ; and Emilio Cardemil
• 刊名：Biochemistry
• 出版年：2002
• 出版时间：October 22, 2002
• 年：2002
• 卷：41
• 期：42
• 页码：12763 - 12770
• 全文大小：218K
• 年卷期：v.41,no.42(October 22, 2002)
• ISSN：1520-4995

文摘

Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase catalyses the reversiblemetal-dependent formation of oxaloacetate and ATP from PEP, ADP, and CO₂ and plays a key role ingluconeogenesis. 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 residuesof the putative Mn²⁺ binding site of the enzyme, were altered. Replacement of these residues by Arg andby Gln, respectively, generated enzymes with 1.9 and 2.8 kcal/mol lower Mn²⁺ binding affinity. LowerPEP binding affinity was inferred for the mutated enzymes from the protection effect of PEP against ureadenaturation. Kinetic studies of the altered enzymes show at least a 5000-fold reduction in V_max for theprimary reaction relative to that for the wild-type enzyme. V_max values for the oxaloacetate decarboxylaseand pyruvate kinase-like activities of PEP carboxykinase were affected to a much lesser extent in themutated enzymes. The mutated enzymes show a decreased steady-state affinity for Mn²⁺ and PEP. Theresults are consistent with Lys²¹³ and His²³³ being at the Mn²⁺ binding site of S. cerevisiae PEPcarboxykinase and the Mn²⁺ affecting the PEP interaction. The different effects of mutations in V_max forthe main reaction and the secondary activities suggest different rate-limiting steps for these reactions.