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 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.
Matharu
A
A
School of Biosciences, University of Wales, Cardiff, UK.
Hayashi
H
H
Kagamiyama
H
H
Maras
B
B
John
R A
RA
D000595:Amino Acid Sequence; D001219:Aspartate Aminotransferases / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D001894:Borohydrides / Q000737:chemistry; D003545:Cysteine / Q000031:analogs & derivatives / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008298:Maleates / Q000378:metabolism*; D018377:Neurotransmitter Agents; D021241:Spectrometry, Mass, Electrospray Ionization; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism
D001894:Borohydrides; D008298:Maleates; D018377:Neurotransmitter Agents; C030272:maleic acid; C009282:sodium cyanoborohydride; D001219:Aspartate Aminotransferases; D014357:Trypsin; D003545:Cysteine; C013461:cysteine sulfinic acid