Lysine 199 is the general acid in the NAD-malic enzyme reaction.
Site-directed mutagenesis was used to change K199 in the Ascaris suum NAD-malic enzyme to A and R and Y126 to F. The K199A mutant enzyme gives a 10(5)-fold decrease in V and a 10(6)-fold decrease in V/K(malate) compared to the WT enzyme. In addition, the ratio for partitioning of the oxalacetate intermediate toward pyruvate and malate changes from a value of 0.4 for the WT enzyme to 1.6 for K199A, and repeating the experiment with A-side NADD gives isotope effects of 3 and 1 for the WT and K199A mutant enzymes, respectively. The K199R mutant enzyme gives only a factor of 10 decrease in V, and the pK for the general acid in this mutant enzyme has increased from 9 for the WT enzyme to >10 for the K199R mutant enzyme. Tritium exchange from solvent into pyruvate is catalyzed by the WT enzyme, but not by the K199A mutant enzyme. The Y126F mutant enzyme gives a 10(3)-fold decrease in V. The oxalacetate partition ratio and isotope effect on oxalacetate reduction for the Y126F mutant enzyme are identical, within error, to those measured for the WT enzyme. Thus, Y126 is important to the overall reaction, but its role at present is unclear. Data are consistent with K199 functioning as the general acid that protonates C3 of enolpyruvate to generate the pyruvate product in the malic enzyme reaction.
Liu
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Department of Chemistry and Biochemistry, University of Oklahoma, 620 Parrington Oval, Norman, Oklahoma 73019, USA.
Karsten
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WE
Cook
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PF
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001120:Arginine / Q000235:genetics; D017165:Ascaris suum / Q000201:enzymology / Q000235:genetics; D002384:Catalysis; D017124:Conserved Sequence; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000737:chemistry* / Q000235:genetics; D008291:Malate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000737:chemistry*; D062907:Oxaloacetic Acid / Q000737:chemistry; D019289:Pyruvic Acid / Q000737:chemistry; D012997:Solvents; D014316:Tritium / Q000737:chemistry
D012997:Solvents; D009243:NAD; D014316:Tritium; D062907:Oxaloacetic Acid; D019289:Pyruvic Acid; D001120:Arginine; D008291:Malate Dehydrogenase; C046446:malate dehydrogenase-(oxaloacetate-decarboxylating) (NAD+); D008239:Lysine; D000409:Alanine