title: Strength of an interloop hydrogen bond determines the kinetic pathway in catalysis by Escherichia coli dihydrofolate reductase.
abstract: On the basis of X-ray crystallographic data, Sawaya and Kraut proposed that Met20 loop conformational changes modulate ligand specificity observed in the catalytic cycle for Escherichia coli dihydrofolate reductase (DHFR) [Sawaya, M. R., and Kraut, J. (1997) Biochemistry 36, 586-603]. Interloop hydrogen bonds stabilize either a closed Met20 loop conformation observed in substrate complexes or an occluded Met20 loop conformation observed in product complexes, respectively. To test this model, we targeted a single hydrogen bond occurring exclusively in the closed Met20 loop conformation. Specifically, Asp122 in the betaF-betaG loop was independently substituted with asparagine, serine, and alanine-amino acids with decreasing abilities to hydrogen-bond. The kinetic analyses of the Asp122 mutants enabled the construction of kinetic schemes at pH 7.0 that demonstrate two striking features. First, a significant correlation exists between decreased binding of nicotinamide adenine dinucleotide phosphate, reduced (NADPH), and decreased hydride transfer rates resulting from these mutations. In other words, the interactions of Asp122 are along the reaction coordinate leading to the transition state. Second, substitutions for Asp122 alter the catalytic pathway preferred by wild-type DHFR under saturating conditions of substrate and cofactor. Overall, the steady-state rate contains contributions from the product off rates from the DHFR.5,6, 7,8-tetrahydrofolate (H4F) and DHFR.NADPH.H4F complexes and from the rate of hydride transfer. These mutational effects support the mechanistic model whereby interloop contacts regulate an equilibrium of Met20 loop conformations that, in turn, modulate ligand affinity and turnover.
authors.affiliation: Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
authors.forename: G P
authors.initials: GP
authors.lastname: Miller
authors.forename: S J
authors.initials: SJ
authors.lastname: Benkovic
mesh_terms: D002384:Catalysis; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology*; D006860:Hydrogen Bonding; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*
chemical_list: D013762:Tetrahydrofolate Dehydrogenase