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

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 However, when Tyr70 of AspAT has been replaced by Phe, the enzyme shows only a faint absorption at 494 nm (epsilon = 522 M-1 cm-1) on the reaction with HOAsp.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 the presence of both aspartate and oxalacetate, and the free energy profiles for the reactions have been similarly obtained. Comparison of the free energy levels in the profiles showed that the interaction of the beta-hydroxy group of HOAsp with the hydroxy group of Tyr70 accounts for 8.7 kJ mol-1 of the 18.5 kJ mol-1 stabilization of the quinonoid intermediate by the beta-hydroxy group. Model building of the active site of AspAT complexed with HOAsp suggests that the rest of the stabilization is mediated through the interaction of the beta-hydroxy group of HOAsp with the protonated epsilon-amino group of Lys258. This interaction is expected to strengthen the hydrogen-bonding network involving Tyr70, HOAsp, and the coenzyme phosphate. A similar network is possibly formed in the carbinolamine intermediate, suggesting ES3 to be the carbinolamine. A mechanism for the reaction of AspAT with HOAsp, which conforms to all the kinetic and spectroscopic data presented here, is proposed. This study provides a basis for subsequent spectroscopic characterization of the HOAsp-AspAT complex, which is a good model for the critical intermediate (quinonoid) structure of the AspAT-catalyzed reactions.