3-DIMENSIONAL STRUCTURE OF A MUTANT ESCHERICHIA-COLI ASPARTATE-AMINOTRANSFERASE WITH INCREASED ENZYMATIC-ACTIVITY

The aspartate and tyrosine aminotransferases from Escherichia coli hav e 43% sequence identity and nearly identical active sites. Both are eq ually good enzymes for dicarboxylate substrates, but the latter transa minates aromatic amino acids 1000 times faster. In an attempt to disco ver the critical residues for this differential substrate specificity, the aspartate aminotransferase mutant V39L has recently been prepared . It showed improved k(cat)/K-m values for aspartate, glutamate and ty rosine and the corresponding oxo acids, mainly due to two to ten times lower K-m values. For example, the K-m values of V39L (wild type) for Asp and Glu are 0.12 (1.0) and 0.85 (2.7) mM respectively. The mutant was co-crystallized with 30 mM maleate from both polyethylene glycol and ammonium sulfate. Both structures were solved and refined to R-fac tors of 0.22 and 0.20 at 2.85 and 2.5 Angstrom resolution respectively . They bear strong resemblance to the closed structure of the wild typ e enzyme complexed with maleate. The unexpected feature is that, for t he first time, the closed form was produced in crystals grown from amm onium sulfate. It is concluded that the mutation has shifted the confo rmational equilibrium towards the closed form, which leads to generall y reduced substrate K(m)s.