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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)
 
 
 
 
 

Acid-Base Chemical Mechanism of Homocitrate Synthase from Saccharomyces cerevisiae.

Homocitrate synthase (acetyl-coenzyme A:2-ketoglutarate C-transferase; E.C. 2.3.3.14) catalyzes the condensation of AcCoA and alpha-ketoglutarate to give homocitrate and CoA. The enzyme was found to be a Zn-containing metalloenzyme using inductively coupled plasma mass spectrometry. Dead-end analogues of alpha-ketoglutarate were used to obtain information on the topography of the alpha-ketoglutarate binding site. The alpha-carboxylate and alpha-oxo groups of alpha-ketoglutarate are required for optimum binding to coordinate to the active site Zn. Optimum positioning of the alpha-carboxylate, alpha-oxo, and gamma-carboxylate of alpha-ketoglutarate is likely mimicked by the location in space of the 2-carboxylate, pyridine nitrogen, and 4 carboxylate of pyridine 2,4-dicarboxylate. The pH dependence of the kinetic parameters was determined to obtain information on the chemical mechanism of homocitrate synthase. The V profile is bell shaped with slopes of 1 and -1, giving pK(a) values of 6.7 and 8.0, while V/K(AcCoA) exhibits a slope of 2 on the acidic side with an average pK(a) value of 6.6 and a slope of -2 on basic side of the profile with an average pK(a) value of 8. 2. The V/K(alpha)(-Kg) pH-rate profile exhibits a single pK(a) of 6.9 on the acidic side and two on the basic side with an average value of 7. 8. The pH dependence of the K(i) for glyoxylate, a competitive inhibitor vs alpha-ketoglutarate, gives a pK(a) of 7.1 for a group, required to be protonated for optimum binding. Data suggest a chemical mechanism for the enzyme in which alpha-ketoglutarate first binds to the active site Zn via its alpha-carboxylate and alpha-oxo groups, followed by acetyl-CoA. A general base then accepts a proton from the methyl of acetyl-CoA, and a general acid protonates the carbonyl of alpha-ketoglutarate in the formation of homocitryl-CoA. The general acid then acts as a base in deprotonating Zn-OH(2) in the hydrolysis of homocitryl-CoA to give homocitrate and CoA. A solvent deuterium kinetic isotope effect of 1 is measured for homocitrate synthase, while a small pH-independent primary kinetic deuterium isotope effect ( approximately 1.3) is observed using deuterioacetyl-CoA. Data suggest rate-limiting condensation to form the alkoxide of homocitryl-CoA, followed by hydrolysis to give products.[1]

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