Ultraviolet difference-spectroscopic studies of substrate and inhibitor binding to Lactobacillus casei dihydrofolate reductase

Abstract

The UV difference spectra generated when methotrexate, trimethoprim or folate bind to L. casei dihydrofolate reductase were analyzed. The difference spectrum produced by methotrexate binding consists of 3 components: 1 closely resembling that observed on protonation of methotrexate, reflecting an increased degree of protonation on binding; a pH-independent contribution corresponding to a 40nm shift to longer wavelengths of a single absorption band of methotrexate; and a component arising from pertubation of tryptophan residue(s) of the enzyme. Quantitative analysis of the pH-dependence of the 1st component shows that the pK of methotrexate is increased from 5.35 to 8.55 (.+-. 0.10) on binding. Folate is not protonated when bound to the enzyme at neutral pH. At pH 7.5, where methotrexate is bound 2000 times more thighly that folate, 1/3 of the difference in binding energy between the 2 compounds arises from the difference in charge state. A similar analysis of the difference spectra generated of trimethoprim binding demonstrates that this compound shows an increase in pK on binding, but only from 7.22 to 7.90 (.+-. 0.10), suggesting that its 2,4-diaminopyrimidine ring does not bid to the enzyme in precisely the same way as the corresponding moiety of methotrexate.