The roles of Tyr(91) and Lys(162) in general acid-base catalysis in the pigeon NADP+-dependent malic enzyme.
The role of general acid-base catalysis in the enzymatic mechanism of NADP+-dependent malic enzyme was examined by detailed steady-state kinetic studies through site-directed mutagenesis of the Tyr(91) and Lys(162) residues in the putative catalytic site of the enzyme. Y91F and K162A mutants showed approx. 200- and 27000-fold decreases in k(cat) values respectively, which could be partially recovered with ammonium chloride. Neither mutant had an effect on the partial dehydrogenase activity of the enzyme. However, both Y91F and K162A mutants caused decreases in the k(cat) values of the partial decarboxylase activity of the enzyme by approx. 14- and 3250-fold respectively. The pH-log(k(cat)) profile of K162A was found to be different from the bell-shaped profile pattern of wild-type enzyme as it lacked a basic pK(a) value. Oxaloacetate, in the presence of NADPH, can be converted by malic enzyme into L-malate by reduction and into enolpyruvate by decarboxylation activities. Compared with wild-type, the K162A mutant preferred oxaloacetate reduction to decarboxylation. These results are consistent with the function of Lys(162) as a general acid that protonates the C-3 of enolpyruvate to form pyruvate. The Tyr(91) residue could form a hydrogen bond with Lys(162) to act as a catalytic dyad that contributes a proton to complete the enol-keto tautomerization.
Kuo
Cheng-Chin
CC
Cardiovascular and Blood Medical Research Center, National Health Research Institutes, Miaoli County, Taiwan, Republic of China.
Lin
Kuan-Yu
KY
Hsu
Yau-Jung
YJ
Lin
Shu-Yu
SY
Lin
Yu-Tsen
YT
Chang
Gu-Gang
GG
Chou
Wei-Yuan
WY
D000143:Acids / Q000737:chemistry; D000468:Alkalies / Q000737:chemistry; D002384:Catalysis; D002942:Circular Dichroism; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D008291:Malate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015394:Molecular Structure; D009154:Mutation / Q000235:genetics; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*
D000143:Acids; D000468:Alkalies; D011994:Recombinant Proteins; D014443:Tyrosine; D009249:NADP; D008291:Malate Dehydrogenase; C031917:malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+); D008239:Lysine