ACCUMULATION OF THE QUINONOID INTERMEDIATE IN THE REACTION CATALYZED BY ASPARTATE-AMINOTRANSFERASE WITH CYSTEINE SULFINIC ACID

The pyridoxal phosphate form of aspartate aminotransferase from Escher ichia coli catalyzes the irreversible conversion of L-cysteine sulfina te to the pyridoxamine phosphate form of the enzyme, bisulfite, and py ruvate, The addition of L-cysteine sulfinate to a solution containing a high concentration of enzyme (approximate to 10 mu M) yields a rapid ly appearing red color (lambda(max) = 520 nm) which decays with a rate constant which is only about 1% of k(cat) (2-3 s(-1) versus 250 s(-1) at 15 degrees C, pH 7), The red color can be assigned to the quinonoi d form of the enzyme substrate complex, which accumulates under these single turnover conditions, The rate of decay of this species is depen dent on that for the decomposition of beta-sulfinylpyruvate (beta-SP), the initial product of the reaction between aspartate aminotransferas e 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 bet a-SP with the pyridoxamine phosphate form of the enzyme, The associati on 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 u nder these conditions when aspartate is substituted for L-cysteine sul finate. (C) 1995 Academic Press, Inc.