CONTRIBUTIONS OF TRYPTOPHAN-24 AND GLUTAMATE-30 TO BINDING LONG-LIVEDWATER-MOLECULES IN THE TERNARY COMPLEX OF HUMAN DIHYDROFOLATE-REDUCTASE WITH METHOTREXATE AND NADPH STUDIED BY SITE-DIRECTED MUTAGENESIS AND NUCLEAR-MAGNETIC-RESONANCE SPECTROSCOPY

Previous NMR studies on the ternary complex of human dihydrofolate red uctase (hDHFR) with methotrexate (MTX) and NADPH detected six long-liv ed bound water molecules. Two of the water molecules, WatA and WatB, s tabilize the structure of the protein while the other four, WatC, WatD , WatE and WatF, are involved in substrate binding and specificity Wat E may also act as a proton shuttle during catalysis. Here, the contrib utions of individual residues to the binding of these water molecules are investigated by performing NMR experiments on ternary complexes of mutant enzymes, W24F, E30A and E30Q. W24 and E30 are conserved residu es that form hydrogen bonds with WatE in crystal structures of DHFR. N uclear Overhauser effects (NOEs) are detected between WatE and the pro tein in all the mutant complexes, hence WatE still has a long lifetime bound to the complex when one of its hydrogen-bonding partners is del eted or altered by mutagenesis. The NOEs for WatE are much weaker, how ever, in the mutants than in wild-type. The NOEs for the other water m olecules in and near the active site, WatA, WatC, WatD and WatF, also tend to be weaker in the mutant complexes. Little or no change is appa rent in the NOEs for WatB, which is located outside the active site, f arthest from the mutated residues. The decreased NOE intensities for t he bound water molecules could be caused by changes in the positions a nd/or lifetimes of the water molecules. Chemical shift and NOE data in dicate that the mutants have structures very similar to that of wild-t ype hDHFR, with possible conformational changes occurring only near th e mutated residues. Based on the lack of structural change in the prot ein and evidence for increased structural fluctuations in the active s ites of the mutant enzymes, it is likely that the NOE changes are caus ed, at least in part, by decreases in the lifetimes of the bound water molecules.