Contributions of the substrate-binding arginine residues to maleate-induced closure of the active site of Escherichia coli aspartate aminotransferase

Crystallography shows that aspartate aminotransferase binds dicarboxylate s ubstrate analogues by bonds to Arg292 and Arg386, respectively [Jager, J, M oser, M. Sauder, U. & Jansonius, J. N. (1994) J. Mol. Biol., 239, 285-305]. The contribution of each interaction to the conformational change that the enzyme undergoes when it binds ligands via these residues, is assessed by probing mutant forms of the enzyme lacking either or both arginines. The pr obes used are NaH3BCN which reduces the cofactor imine, the reactive substr ate analogue, cysteine sulfinate and proteolysis by trypsin. The unreactive substrate analogue, maleate, is used to induce closure. Each single mutant reacted only 2.5-fold more slowly with NaH3BCN than the wild-type indicati ng that charge repulsion by the arginines contributes little to maintaining the open conformation. Maleate lowered the rate of reduction of the wild-t ype enzyme more than 300-fold but had little effect on the reaction of the mutant enzymes indicating that the ability of this dicarboxylate analogue t o bridge the arginines precisely makes the major contribution to closure. T he R292L mutant reacted 20 times more rapidly with cysteine sulfinate than R386L but 5 x 10(4) times more slowly than the wild-type enzyme, consistent with the proposal that enzyme's catalytic abilities are not developed unle ss closure is induced by bridging of the arginines. Proteolysis of the muta nts with trypsin showed that, in the wild-type enzyme, the bonds most susce ptible to trypsin are those contributed by Arg292 and Arg386. Proteolysis o f the next most susceptible bond, at Arg25 in the double mutant, was protec ted by maleate demonstrating the presence of an additional site on the enzy me for binding dicarboxylates.