Determinants of performance in the isocitrate dehydrogenase of Escherichia coli.
The substrate specificity of the NADP-dependent isocitrate dehydrogenase of Escherichia coli was investigated by combining site-directed mutagenesis and utilization of alternative substrates. A comparison of the kinetics of the wild-type enzyme with 2R-malate reveals that the gamma-carboxylate of 2R,3S-isocitrate contributes a factor of 12,000,000 to enzyme performance. Analysis of kinetic data compiled for 10 enzymes and nine different substrates reveals that a factor of 1,650 can be ascribed to the hydrogen bond formed between S113 and the gamma-carboxylate of bound isocitrate, a factor of 150 to the negative charge of the gamma-carboxylate, and a factor of 50 for the gamma-methyl. These results are entirely consistent with X-ray structures of Michaelis complexes that show a hydrogen bond positions the gamma-carboxylate of isocitrate so that a salt bridge can form to the nicotinamide ring of NADP.
Dean
A M
AM
Department of Biological Chemistry, Chicago Medical School, Illinois 60064-3095, USA.
Shiau
A K
AK
Koshland
D E
DE
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006860:Hydrogen Bonding; D007521:Isocitrate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007523:Isocitrates / Q000378:metabolism; D007700:Kinetics; D008293:Malates / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D013379:Substrate Specificity; D013644:Tartrates / Q000378:metabolism
D001426:Bacterial Proteins; D007523:Isocitrates; D008293:Malates; D013644:Tartrates; D009249:NADP; C034219:isocitric acid; D007521:Isocitrate Dehydrogenase; C029768:tartaric acid