A HYDROGEN-BONDING NETWORK MODULATING ENZYME FUNCTION - ASPARAGINE-194 AND TYROSINE-225 OF ESCHERICHIA-COLI ASPARTATE-AMINOTRANSFERASE

The electron distribution within the coenzyme or coenzyme-substrate conjugate needs to be properly regulated during the catalytic process of aspartate aminotransferase (AspAT). Asn194 and Tyr225 may function in regulating the electron distribution through hydrogen-bonding to O(3') of the coenzyme, pyridoxal 5'-phosphate (PLP) or pyridoxamine 5'-phosphate (PMP). The roles of Tyr225 have already been explored by site-directed mutagenesis (Inoue et al., 1991; Goldberg et al., 1991). In the present studies, the mutant enzymes Asn194-->Ala and Asn194-->Ala + Tyr225-->Phe were analyzed kinetically and spectroscopically and were compared with the wild-type and Tyr225-Phe enzymes. The kinetic studies showed that Asn 194 is not essential for AspAT catalysis, although the K(d) values for the substrates were increased by 10- to 50-fold upon the replacement of Asn194. The measurements of the absorption and fluorescence excitation spectra revealed that the ratio of an enolimine to a ketoenamine form was considerably increased as a tautomeric form of the protonated PLP in the active site of the double mutant enzyme. The pH-pK(d) relationship for the binding of maleate to AspAT could be explained by a simple thermodynamic cycle where only one ionizing group (the imine nitrogen of the internal aldimine bond) affects the binding of maleate. The analyses of the pH-pK(d) curves for the wild-type and mutant enzymes showed that (i) the hydrogen bond between O(3') of PLP and Asn 194 is weakened by the binding of maleate to AspAT, while the hydrogen bond between O(3') and Tyr225 is not changed, and that (ii) the replacement of Asn194 causes some effect hampering the binding of maleate. Asn194 is also situated within hydrogen-bonding bonding distance to the alpha-carboxylate group of the substrate and Arg386 which is known to function in substrate binding through the interaction with the substrate alpha-carboxylate. The above findings were discussed in terms of the hydrogen-bonding network: Tyr225 --> O(3') of the coenzyme <-- Asn194 <-- Arg386 <--carboxylate group of the substrate (analogue). We propose the idea that Asn194 lowers the pK(a) of the imine nitrogen of the internal aldimine bond to facilitate a transaldimination step and that Asn 194 functions in substrate binding through hydrogen-bonding to the substrate carboxylate and/or holding the side chain of Arg386 in a position suitable for substrate binding. After formation of the coenzyme-substrate conjugate (an external aldimine intermediate), Tyr225 would be the main residue that regulates the electron distribution through hydrogen-bonding to O(3') of the coenzyme.