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Characterization of the functional role of Asp141, Asp194, and Asp464 residues in the Mn2+-l-malate binding of pigeon liver malic enzyme

Published online by Cambridge University Press:  01 February 2000

WEI-YUAN CHOU
Affiliation:
Department of Biochemistry, National Defense Medical Center, Taipei 100, Taiwan, Republic of China
HWEI-PING CHANG
Affiliation:
Department of Biochemistry, National Defense Medical Center, Taipei 100, Taiwan, Republic of China
CHIEN-HSIUN HUANG
Affiliation:
Department of Biochemistry, National Defense Medical Center, Taipei 100, Taiwan, Republic of China
CHENG-CHIN KUO
Affiliation:
Department of Biochemistry, National Defense Medical Center, Taipei 100, Taiwan, Republic of China
LIANG TONG
Affiliation:
Department of Biological Sciences, Columbia University, New York, New York 10027
GU-GANG CHANG
Affiliation:
Department of Biochemistry, National Defense Medical Center, Taipei 100, Taiwan, Republic of China
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Abstract

Pigeon liver malic enzyme was inactivated and cleaved at Asp141, Asp194, and Asp464 by the Cu2+-ascorbate system in acidic environment. Site-specific mutagenesis was performed at these putative metal-binding sites. Three point mutants, D141N, D194N, and D464N; three double mutants, D(141,194)N, D(194,464)N, and D(141,464)N; and a triple mutant, D(141,194,464)N; as well as the wild-type malic enzyme (WT) were successfully cloned and expressed in Escherichia coli cells. All recombinant enzymes, except the triple mutant, were purified to apparent homogeneity by successive Q-Sepharose and adenosine-2′,5′-bisphosphate-agarose columns. The mutants showed similar apparent Km,NADP values to that of the WT. The Km,Mal value was increased in the D141N and D194N mutants. The Km,Mn value, on the other hand, was increased only in the D141N mutant by 14-fold, corresponding to ∼1.6 kcal/mol for the Asp141-Mn2+ binding energy. Substrate inhibition by l-malate was only observed in WT, D464N, and D(141,464)N. Initial velocity experiments were performed to derive the various kinetic parameters. The possible interactions between Asp141, Asp194, and Asp464 were analyzed by the double-mutation cycles and triple-mutation box. There are synergistic weakening interactions between Asp141 and Asp194 in the metal binding that impel the D(141,194)N double mutant to an overall specificity constant [kcat/(Kd,MnKm,MalKm,NADP)] at least four orders of magnitude smaller than the WT value. This difference corresponds to an increase of 6.38 kcal/mol energy barrier for the catalytic efficiency. Mutation at Asp464, on the other hand, has partial additivity on the mutations at Asp141 and Asp194. The overall specificity constants for the double mutants D(194,464)N and D(141,464)N or the triple mutant D(141,194,464)N were decreased by only 10- to 100-fold compared to the WT. These results strongly suggest the involvement of Asp141 in the Mn2+-l-malate binding for the pigeon liver malic enzyme. The Asp194 and Asp464, which may be oxidized by nonspecific binding of Cu2+, are involved in the Mn2+-l-malate binding or catalysis indirectly by modulating the binding affinity of Asp141 with the Mn2+.

Type
Research Article
Information
Protein Science , Volume 9 , Issue 2 , February 2000 , pp. 242 - 251
Copyright
© 2000 The Protein Society

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Characterization of the functional role of Asp141, Asp194, and Asp464 residues in the Mn2+-l-malate binding of pigeon liver malic enzyme
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Characterization of the functional role of Asp141, Asp194, and Asp464 residues in the Mn2+-l-malate binding of pigeon liver malic enzyme
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Characterization of the functional role of Asp141, Asp194, and Asp464 residues in the Mn2+-l-malate binding of pigeon liver malic enzyme
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