ASYMMETRIC ALLOSTERIC ACTIVATION OF ESCHERICHIA-COLI GLUCOSAMINE-6-PHOSPHATE DEAMINASE PRODUCED BY REPLACEMENTS OF TYR-121

Tyrosine 121, a residue located in a alpha-helical polypeptide segment of glucosamine 6-phosphate deaminase from Escherichia coli, has recen tly been proposed to have a role in the binding of the allosteric acti vator N-acetyl-D-glucosamine B-phosphate. Accordingly, the site-direct ed mutants Tyr 121-Thr and Tyr 121-Trp were constructed, to assess exp erimentally the role of Tyr 121 in the allosteric function of the enzy me. The kinetic study of both mutant forms revealed that the replaceme nts caused striking changes in allosteric activator binding and allost eric properties, when compared to the wild-type enzyme. While the wild -type deaminase behaves as a classical allosteric K-system which can b e described by the allosteric concerted model, both mutant forms prese nt an asymmetric behavior toward the allosteric activator, which can b e described as two distinct half-of-the-sites allosteric activation st eps occurring with different affinities for the N-acetyl-D-glucosamine 6-phosphate. During the first (high affinity) activation phase, the m utant forms of deaminase behave as mixed K/V allosteric enzyme. The bi phasic activation curve was also demonstrated by direct binding measur ements of the C-14-labeled activator to Tyr 121-Trp and Tyr 121-Thr de aminases. The kinetic analysis of these mutant forms also showed that the threonine replacement produced an important distortion of the enzy me structure reflected in a considerable decrease of its catalytic eff iciency. This finding suggests that the Thr replacement at position 12 1 produces structural perturbations which are absent in the Tyr 121-Tr p mutant form of the enzyme, thus emphasizing the structural importanc e of interactions at this position between the helix and the protein c ore.