The function of amino acid residues contacting the nicotinamide ring of NADPH in dihydrofolate reductase from Escherichia coli.
The importance of three amino acid residues contacting the nicotinamide ring of NADPH in Escherichia coli dihydrofolate reductase has been defined using site-directed mutagenesis and detailed steady-state and pre-steady-state kinetic experiments. Replacement of Tyr-100 with either glycine or isoleucine (Y100G or Y100I) disrupts an aromatic-aromatic interaction between the phenolic side chain and the nicotinamide ring. Both mutations remove the differential binding of the oxidized and reduced coenzymes implicating Tyr-100 as a major determinant for coenzyme specificity. Replacement of Ser-49 for alanine (S49A), designed to either displace or reduce the polarizability of a bound water molecule contacting the N1 of the nicotinamide ring, affects only the rate of release of NADP+. Replacement of Ile-14 with alanine (I14A), designed to alter both a weakly polar and a hydrogen bonding interaction with the periphery of the nicotinamide ring, affects only the binding of NADPH. Y100I, Y100G, and I14A all increase the activation barrier for the chemical step by approximately 2 kcal/mol. The lack of an effect for S49A suggests that water structure is not important for stabilizing the hydride transfer transition state. In addition, the nominal effects observed for these mutations disfavor the hypothesis that neighboring amino acid residues participate in the stabilization of the reaction transition state through polar or weakly polar contacts.
Adams
J A
JA
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
Fierke
C A
CA
Benkovic
S J
SJ
D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology*; D005493:Folic Acid Antagonists; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009249:NADP / Q000737:chemistry*; D009536:Niacinamide / Q000737:chemistry*; D011485:Protein Binding; D011621:Pteridines / Q000494:pharmacology; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics; D013816:Thermodynamics
D000596:Amino Acids; D005493:Folic Acid Antagonists; D011621:Pteridines; D009536:Niacinamide; D009249:NADP; D013762:Tetrahydrofolate Dehydrogenase