The overall objective of the study is to understand the interplay between protein structure and mechanism, for some biologically important macromolecules. As a model system the enzyme dihydropteridine reductase (DHPR) was studied. DHPR catalyses the NADH-mediated reduction of quinonoid dihydrobiopterin to give tetrahydrobiopterin, which functions as an essential cofactor in the biosynthetic reactions that convert phenylalanine to tyrosine, tyrosine to dihydroxyphenylalanine, and tryptophan to dihydroxytrptophan. In this study the fluorescence signature of tyrosine, tryptophan, NADH, NAD+, some pteridines and the enzyme dihydropteridine reductase (DHPR) was determined. The presence of DHPR quenches the fluorescence of the cofactor, NADH, and the inhibitors folic acid, methotrexate and trimethoprim. One intriguing result was that for one inhibitor, 6,7-dimethyl 5,6,7,8 tetrahydropterin, the presence DHPR results in an enhancement of fluorescence. We also measured the fluorescence spectra of the ternary complexes involving DHPR/NADH/inhibitor. It is clear from our results that small molecules interact with DHPR and it appears that the inhibitors interact with DHPR even in the absence of the cofactor, NADH. We also conclude that fluorescence spectroscopy is an effective tool to study DHPR and we will extend this work to do fluorescence based transient measurements. Rosa Rosales is a participant in the QCC-NIH Bridges to the Baccalaureate Program (grant 1 R25 GM65096-03).
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