Accumulation of the quinonoid intermediate in the reaction catalyzed by aspartate aminotransferase with cysteine sulfinic acid.
The pyridoxal phosphate form of aspartate aminotransferase from Escherichia coli catalyzes the irreversible conversion of L-cysteine sulfinate to the pyridoxamine phosphate form of the enzyme, bisulfite, and pyruvate. The addition of L-cysteine sulfinate to a solution containing a high concentration of enzyme (approximately 10 microM) yields a rapidly appearing red color (lambda max = 520 nm) which decays with a rate constant which is only about 1% of kcat (2-3 s-1 versus 250 s-1 at 15 degrees C, pH 7). The red color can be assigned to the quinonoid form of the enzyme substrate complex, which accumulates under these single turnover conditions. The rate of decay of this species is dependent on that for the decomposition of beta-sulfinylpyruvate (beta-SP), the initial product of the reaction between aspartate aminotransferase and L-cysteine sulfinate. Trapping beta-SP with morpholine or malate dehydrogenase plus NADH abolishes the transient red color; therefore, the intermediate accumulates by virtue of the reverse reaction of beta-SP with the pyridoxamine phosphate form of the enzyme. The association and dissociation rate constants of beta-SP with the pyridoxamine-5'-phosphate form of the enzyme are 2 x 10(7) M-1 s-1 and 400 s-1, respectively, at 15 degrees C. No red transient species is observed under these conditions when aspartate is substituted for L-cysteine sulfinate.
Furumo
N C
NC
Department of Molecular and Cell Biology, University of California, Berkeley 94720, USA.
Kirsch
J F
JF
D001219:Aspartate Aminotransferases / Q000378:metabolism*; D003545:Cysteine / Q000031:analogs & derivatives / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D006207:Half-Life; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007700:Kinetics; D008954:Models, Biological; D018377:Neurotransmitter Agents; D011733:Pyridoxamine / Q000031:analogs & derivatives / Q000378:metabolism; D011809:Quinones / Q000378:metabolism*; D013053:Spectrophotometry
D007202:Indicators and Reagents; D018377:Neurotransmitter Agents; D011809:Quinones; D011733:Pyridoxamine; D001219:Aspartate Aminotransferases; D003545:Cysteine; C010627:pyridoxamine phosphate; C013461:cysteine sulfinic acid