SIGNIFICANT IMPROVEMENT TO THE CATALYTIC PROPERTIES OF ASPARTATE-AMINOTRANSFERASE - ROLE OF HYDROPHOBIC AND CHARGED RESIDUES IN THE SUBSTRATE-BINDING POCKET

The substrate specificity of tyrosine aminotransferase (eTAT) from Esc herichia coli has been tested by transferring the critically different residues Leu39, Glu141, and Arg293 into equivalent positions of aspar tate aminotransferase (eAAT). These residues are not directly involved in the catalytic process. The single mutant eAAT V39L possesses great er values of k(cat)/K(M) not only for tyrosine but also for aspartate and glutamate. In contrast, the double mutant eAAT P141E,A293R and als o the triple mutant eAAT V39L,P141E,A293R exhibit smaller changes of k (cat)/K(M). The converse mutants of tyrosine aminotransferase, in whic h critical residues of eAAT (Val39) and of mitochondrial AAT (Ala39, V al37) were transferred into equivalent positions of eTAT, exhibited ge nerally decreased values of k(cat)/K(M) for both dicarboxylic and arom atic substrates. On the basis of the known structures of eAAT and eAAT V39L as well as of a refined model of eTAT, these results indicate th at the different substrate specificities of eAAT and eTAT are due to m ultiple side chain differences and minor rearrangements of the backbon e. The generally improved catalytic efficiency of the mutant eAAT V39L appears to be due to an indirect effect, namely, the facilitated clos ure of the active site upon substrate binding.