SUBSTITUTION OF APOLAR RESIDUES IN THE ACTIVE-SITE OF ASPARTATE-AMINOTRANSFERASE BY HISTIDINE - EFFECTS ON REACTION AND SUBSTRATE-SPECIFICITY

In an attempt to change the reaction and substrate specificity of aspa rtate aminotransferase, several apolar active-site residues were subst ituted in turn with a histidine residue. Aspartate aminotransferase W1 40H (of Escherichia coli) racemizes alanine seven times faster (k'(cat ) = 2.2 X 10(-4) s(-1)) than the wild-type enzyme, while the aminotran sferase activity toward L-alanine was sixfold decreased. X-ray crystal lographic analysis showed that the structural changes brought about by the mutation are limited to the immediate environment of H140. In con trast to the tryptophan side chain in the wild-type structure, the imi dazole ring of H140 does not form a stacking interaction with the coen zyme pyridine ring. The angle between the two ring planes is about 50 degrees. Pyridoxamine 5'-phosphate dissociates 50 times more rapidly f rom the W140H mutant than from the wild-type enzyme. A model of the st ructure of the quinonoid enzyme substrate intermediate indicates that H140 might assist in the reprotonation of C alpha of the amino acid su bstrate from the re side of the deprotonated coenzyme-substrate adduct in competition with si-side reprotonation by K258. In aspartate amino transferase I17H (of chicken mitochondria), the substituted residue al so lies on the re side of the coenzyme. This mutant enzyme slowly deca rboxylates L-aspartate to L-alanine (k'(cat) = 8 X 10(-5) s(-1)). No b eta-decarboxylase activity is detectable in the wild-type enzyme. In a spartate aminotransferase V37H (of chicken mitochondria), the mutated residue lies besides the coenzyme in the plane of the pyridine ring; n o change in reaction specificity was observed. All three mu tations, i .e. W140-->H, I17-->H and V37-->H, decreased the aminotransferase acti vity toward aromatic amino acids by 10-100-fold, while decreasing the activity toward dicarboxylic substrates only moderately to 20%, 20% an d 60% of the activity of the wild-type enzymes, respectively. In all t hree mutant enzymes, the decrease in aspartate aminotransferase activi ty at pH values lower than 6.5 was more pronounced than in the wild-ty pe enzyme, apparently due to the protonation of the newly introduced h istidine residues. The study shows that substitutions of single active -site residues may result in altered reaction and substrate specificit ies of pyridoxal-5'-phosphate-dependent enzymes.