Evidence for a critical glutamyl and an aspartyl residue in the function of pig heart diphosphopyridine nucleotide dependent isocitrate dehydrogenase.
The pH dependence of the maximum velocity of the reaction catalyzed by diphosphopyridine nucleotide (DPN) dependent isocitrate dehydrogenase 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 to 33 degrees C, suggesting the ionization of a carboxyl rather than an imidazolium ion. The enzyme is inactivated upon incubation with 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide in the presence of glycinamide or glycine ethyl ester. This inactivation is dependent on pH and the rate constant (k) increases as the pH is decreased in the range 7.3 to 6.25. A plot of 1/(H+) vs. 1/k suggests that the enzyme is inactivated as a result of the modification of a single ionizable group in this pH range. The coenzyme DPN and substrate alpha-ketoglutarate do not affect the rate of inactivation. In contrast, manganous ion (2 mM) and isocitrate (60 mM) produce a sevenfold decrease in the rate constant. The allosteric activator ADP (1 mM) does not itself influence the rate of inactivation; however, 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]glycine ethyl ester show a net incorporation of 2 mol of glycine ethyl ester per 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 two major radioactive spots with approximately 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 manganous ion and isocitrate results in significant reduction in the incorporation of radioactivity into the two dipeptides. These results suggest that carbodiimide attacks one glutamyl and one aspartyl residue per subunit of the enzyme and that the integrity of these residues is crucial for the enzymatic activity.
Evidence for a critical glutamyl and an aspartyl residue in the function of pig heart diphosphopyridine nucleotide dependent isocitrate dehydrogenase. The pH dependence of the maximum velocity of the reaction catalyzed by diphosphopyridine nucleotide (DPN) dependent isocitrate dehydrogenase 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 to 33 degrees C, suggesting the ionization of a carboxyl rather than an imidazolium ion. The enzyme is inactivated upon incubation with 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide in the presence of glycinamide or glycine ethyl ester. This inactivation is dependent on pH and the rate constant (k) increases as the pH is decreased in the range 7.3 to 6.25. A plot of 1/(H+) vs. 1/k suggests that the enzyme is inactivated as a result of the modification of a single ionizable group in this pH range. The coenzyme DPN and substrate alpha-ketoglutarate do not affect the rate of inactivation. In contrast, manganous ion (2 mM) and isocitrate (60 mM) produce a sevenfold decrease in the rate constant. The allosteric activator ADP (1 mM) does not itself influence the rate of inactivation; however, 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]glycine ethyl ester show a net incorporation of 2 mol of glycine ethyl ester per 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 two major radioactive spots with approximately 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 manganous ion and isocitrate results in significant reduction in the incorporation of radioactivity into the two dipeptides. These results suggest that carbodiimide attacks one glutamyl and one aspartyl residue per subunit of the enzyme and that the integrity of these residues is crucial for the enzymatic activity.