"Catch 222": Effects of Symmetry on Binding and Catalysis in R67 Dihydrofolate Reductase

University Of Tennessee Knoxville, Knoxville TN

Investigators

Abstract

Dihydrofolate reductase (DHFR) is an important enzyme in cellular metabolism. One variant of the enzyme, R67 DHFR, which is found in some strains of the bacterium Escherichia coli, is unrelated genetically and structurally to chromosomal DHFR. R67 DHFR is a homotetramer; its structure resembles a doughnut and the central hole describes the (single) active site. This enzyme possesses exact 222 symmetry, requiring that each binding site must have 3 symmetry related sites. Since two different ligands are used in the reaction, substrate (dihydrofolate) and cofactor (NADPH), there must be an unusual approach towards catalysis. For example, binding studies indicate only two molecules bind concurrently; the combinations are two substrates or two cofactors or one substrate plus one cofactor. Only the latter results in catalysis. Thus binding of cofactor and substrate cannot be independently optimized. This is just one example suggesting that R67 DHFR is a primitive enzyme. To probe how this simple enzyme works, binding studies of substrate and cofactor analogs will monitor important interactions in the ground state and temperature dependence studies of catalysis will explore the transition state. In addition, the active site symmetry will be broken using a quadruplicated gene construct. Several asymmetric mutants have already been constructed, the next step is to "mix and match" the mutations to allow independent manipulation of each half of the active site pore. This should lead to favorable interactions with NADPH on one side of the pore and complementary interactions with dihydrofolate on the other and perhaps result in a more efficient enzyme. The broader impacts of this research are diverse. The PI has an established a strong track record in engaging undergraduates in research. Several undergraduates, including underrepresented minority students, participate in R67 DHFR research each year. Some undergraduate students have been co-authors on publications. In this project, graduate students, including a hearing-impaired student, will receive rigorous training in various biochemical disciplines. In addition, collaborations with computational biologist and a statistician provide opportunities for interdisciplinary training of students. The programs that are developed as a part of the collaboration will be available to others via Internet.