Reaction of aspartate aminotransferase with L-erythro-3-hydroxyaspartate: involvement of Tyr70 in stabilization of the catalytic intermediates

H Hayashi, H Kagamiyama


The reaction of Escherichia coli aspartate aminotransferase (AspAT) with L-erythro-3-hydroxyaspartate (HOAsp) produces an intense absorption at 494 nm (epsilon = 13,650 M-1 cm-1), which is ascribed to the quinonoid intermediate. However, when Tyr70 of AspAT has been replaced by Phe, the enzyme shows only a faint absorption at 494 nm (epsilon = 522 M-1 cm-1) on the reaction with HOAsp. This indicates the involvement of the hydroxy group of Tyr70 in stabilizing the quinonoid intermediate formed from HOAsp and pyridoxal 5'-phosphate at the AspAT active site. Kinetic analysis of the absorption changes of the wild-type and Y70F mutant AspATs has shown that the reactions with HOAsp conform to the equation, EL + S<-->ES1<-->ES2<-->ES3<-->EM + P, in which there is a rapid formation of the quinonoid intermediate (ES2) from ES1, followed by a slow equilibrium between ES2 and ES3. ES3 absorbs primarily at 330 nm. The kinetic parameters for individual steps have been determined, and free energy profiles for the reactions of the two enzymes with HOAsp have been obtained. The stability of the quinonoid intermediates of the two enzymes in the normal catalytic reactions with aspartate has been assessed by static measurement of the spectra in t...Continue Reading


May 1, 1978·Archives of Biochemistry and Biophysics·R C Harruff, W T Jenkins
Jan 1, 1990·Annals of the New York Academy of Sciences·J E TaylorA Arnone
Apr 15, 1984·Journal of Molecular Biology·J F KirschP Christen

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