Evidence for a critical glutamyl and an aspartyl residue in the function of pig heart diphosphopyridine nucleotide dependent isocitrate dehydrogenase

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Abstract
The pH dependence of the Vmax of the reaction catalyzed by diphosphopyridine nucleotide (DPN) dependent isocitrate dehydrogenase [EC 1.1.1.41] indicates the requirement for the basic form of an ionizable group in the enzyme-substrate complex with a pK of 6.6. This pK is unaltered from 10-33.degree. C, suggesting the ioninzation of a carboxyl rather than an imidazolium ion. The enzyme is inactivated on incubation with 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide in the presence of glycinamide or glucine ethyl ester. This inactivation is dependent on pH and the rate constant (k) increases as the pH is decreased in the range 7.3-6.25. A plot of 1/ [H+ concentration] vs. 1/k suggests that the enzyme is inactivated as a result of the modification of 1 ionizable group in this pH range. The coenzyme DPN and substrate .alpha.-ketoglutarate do not affect the rate of inactivation. In contrast, Mn2+ (2 mM) and isocitrate (60 mM) produce a 7-fold decrease in the rate constant. The allosteric activator ADP (1 mM) does not itself influence the rate of inactivation; but it reduces the concentration of Mn2+ (1 mM) and isocitrate (20 mM) required to produce the same decrease in the inactivation constant. These observations imply that the modification occurs at the substrate-binding site. Experiments employing [1-14C]glucine ethyl ester show a net incorporation of 2 mol of glycine ethyl ester/subunit (40,000), concomitant with the complete inactivation of the enzyme. The radioactive modified enzyme, after removal of excess reagent by dialysis, was exhaustively digested with proteolytic enzymes. High voltage electrophoretic analyses of the hydrolysate at pH 6.4 and 3.5 yield 2 major radioactive spots with aporoximately equal intensity, which correspond to .gamma.-glutamylglycine and .beta.-aspartylglycine, the ultimate products of reaction with glutamic and aspartic acids, respectively. Modification in the presence of Mn2 + and isocitrate results in significant reduction in the incorporation of radioactivity into the 2 dipeptides. Carbodiimide may attack 1 glutamyl and 1 aspartyl residue/subunit of the enzyme. The integrity of these residues is probably crucial for the enzymatic activity.
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