PROBES OF LIGAND-INDUCED CONFORMATIONAL CHANGE IN ASPARTATE-AMINOTRANSFERASE

Sodium borohydride and sodium cyanoborohydride were assessed as potent ial reagents for determining ligand-induced changes in accessibility t o the active-site of aspartate aminotransferase. Rates of reduction of the imine formed between Lys258 and pyridoxal phosphate were determin ed in the presence of increasing concentrations of the dicarboxylate s ubstrate analogues glutarate and maleate. The rate of reduction decrea sed to a limiting value which was about 40-fold lower than the equival ent rate in the absence of dicarboxylate. Analysis of the reaction was complicated by the increasing protonation of the imine which accompan ied binding of dicarboxylates. Allowing for this increase, the true de crease in accessibility to NaBH3CN was estimated to be approximately 4 00-fold. Arguments are presented in support of a proposal that the rat io of closed to open conformer of the dicarboxylate-liganded enzyme is approximately 150. The effects of increasing ligand concentration on the reactivity of Cys390 were found to take place in the same range as was observed for NaBH3CN reduction. Conversely, very much higher conc entrations of the dicarboxylates were required to protect against prot eolysis by trypsin. It is concluded that NaBH3CN reduction and reactiv ity of cysteine are good determinants of the conformational status of the enzyme but that resistance to tryptic digestion is due to an addit ional binding mode for the dicarboxylates.