SIGNIFICANT IMPROVEMENT TO THE CATALYTIC PROPERTIES OF ASPARTATE-AMINOTRANSFERASE - ROLE OF HYDROPHOBIC AND CHARGED RESIDUES IN THE SUBSTRATE-BINDING POCKET
E. Kohler et al., SIGNIFICANT IMPROVEMENT TO THE CATALYTIC PROPERTIES OF ASPARTATE-AMINOTRANSFERASE - ROLE OF HYDROPHOBIC AND CHARGED RESIDUES IN THE SUBSTRATE-BINDING POCKET, Biochemistry, 33(1), 1994, pp. 90-97
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.