CONTRIBUTION OF A SALT BRIDGE TO BINDING-AFFINITY AND DUMP ORIENTATION TO CATALYTIC RATE - MUTATION OF A SUBSTRATE-BINDING ARGININE IN THYMIDYLATE SYNTHASE

Invariant arginine 179, one of four arginines that are conserved in al l thymidylate synthases (TS) and that bind the phosphate moiety of the substrate 2'-deoxyuridine-5'-monophosphate (dUMP), can he altered eve n to a negatively charged glutamic acid with little effect on k(cat). In the mutant structures, ordered water or the other phosphate-binding arginines compensate for the hydrogen bonds made by Arg179 in the wil d-type enzyme and there is almost no change in the conformation or bin ding site of dUMP, Correlation of dUMP K(d)s for TS R179A and TS R179K with the structures of their binary complexes shows that the positive charge on Arg179 contributes significantly to dUMP binding affinity, k(cat)/K-m for dUMP measures the rate of dUMP binding to TS during the ordered bi-substrate reaction, and in the ternary complex dUMP provid es a binding surface for the cofactor. k(cat)/K-m reflects the ability of the enzyme to accept a properly oriented dUMP for catalysis and is less sensitive than is K-d to the changes in electrostatics at the ph osphate binding site.