maleic-acid and sodium-cyanoborohydride

Crystallography shows that aspartate aminotransferase binds dicarboxylate substrate analogues by bonds to Arg292 and Arg386, respectively [Jager, J, Moser, 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 probes used are NaH(3)BCN which reduces the cofactor imine, the reactive substrate 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 NaH(3)BCN than the wild-type indicating that charge repulsion by the arginines contributes little to maintaining the open conformation. Maleate lowered the rate of reduction of the wild-type enzyme more than 300-fold but had little effect on the reaction of the mutant enzymes indicating that the ability of this dicarboxylate analogue to bridge the arginines precisely makes the major contribution to closure. The 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 unless closure is induced by bridging of the arginines. Proteolysis of the mutants with trypsin showed that, in the wild-type enzyme, the bonds most susceptible to trypsin are those contributed by Arg292 and Arg386. Proteolysis of the next most susceptible bond, at Arg25 in the double mutant, was protected by maleate demonstrating the presence of an additional site on the enzyme for binding dicarboxylates.

Topics: Amino Acid Sequence; Aspartate Aminotransferases; Binding Sites; Borohydrides; Cysteine; Escherichia coli; Hydrogen-Ion Concentration; Kinetics; Maleates; Neurotransmitter Agents; Spectrometry, Mass, Electrospray Ionization; Spectrophotometry, Ultraviolet; Substrate Specificity; Trypsin

Sodium borohydride and sodium cyanoborohydride were assessed as potential reagents for determining ligand-induced changes in accessibility to the active-site of aspartate aminotransferase. Rates of reduction of the imine formed between Lys258 and pyridoxal phosphate were determined in the presence of increasing concentrations of the dicarboxylate substrate analogues glutarate and maleate. The rate of reduction decreased to a limiting value which was about 40-fold lower than the equivalent rate in the absence of dicarboxylate. Analysis of the reaction was complicated by the increasing protonation of the imine which accompanied binding of dicarboxylates. Allowing for this increase, the true decrease in accessibility to NaBH3CN was estimated to be approximately 400-fold. Arguments are presented in support of a proposal that the ratio 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 concentrations of the dicarboxylates were required to protect against proteolysis by trypsin. It is concluded that NaBH3CN reduction and reactivity of cysteine are good determinants of the conformational status of the enzyme but that resistance to tryptic digestion is due to an additional binding mode for the dicarboxylates.

Topics: Animals; Aspartate Aminotransferases; Borohydrides; Cysteine; Glutarates; Hydrolysis; Kinetics; Ligands; Maleates; Molecular Probes; Myocardium; Oxidation-Reduction; Protein Conformation; Swine