Affinity labeling of oxaloacetate decarboxylase by novel dichlorotriazine linked alpha-ketoacids

The 4-aminophenyloxanilic acid and beta-mercaptopyruvic acid linked to the reactive diclorotriazine ring, were studied as active site-direct affinity labels towards oxaloacetate decarboxylase (EC 4.1.1.3, OXAD). Oxaloacetate decarboxylase when incubated with 4-aminophenyloxanilic-diclorotriazine (AP OD) or beta-mercaptopyruvic-diclorotriazine (MPD) at pH 7.0 and 25 degrees C shows a time-dependent and concentration-dependent loss of enzyme activit y. The inhibition was irreversible and activity cannot be recovered either by extensive dialysis or gel-filtration chromatography. The enzyme inactiva tion following the Kitz & Wilson kinetics for time-dependent irreversible i nhibition. The observed rate of enzyme inactivation (k(obs)) exhibits a non -linear dependence on APOD or MPD concentration with maximum rate of inacti vation (k(3)) of 0.013 min(-1) and 0.0046 min(-1) and K-D equal to 20.3 and 156 mu M respectively. The inactivation of oxaloacetate decarboxylase by A POD and MPD is competitively inhibited by OXAD substrate and inhibitors, su ch as oxaloacetate, ADP and oxalic acid whereas Mn+2 enhances the rate of i nactivation. The rate of inactivation of OXAD by APOD shows a pH dependence with an inflection point at 6.8, indicating a possible histidine derivatiz ation by the label. These results show that APOD and MPD demonstrate the ch aracteristics of an active-site probe towards the oxaloacetate binding site of oxaloacetate decarboxylase.