Dihydrofolate reductase from Escherichia coli: probing the role of aspartate-27 and phenylalanine-137 in enzyme conformation and the binding of NADPH.
In the absence of ligands, dihydrofolate reductase from Escherichia coli exists in at least two interconvertible conformations, only one of which binds NADPH with high affinity. This equilibrium is pH dependent, involving an ionizable group of the enzyme (pK approximately 5.5), and the proportion of the NADPH-binding conformer increases from 42% at pH 5 to 65% at pH 8. The role of specific amino acids in enzyme conformation has been investigated by studying the kinetics of NADPH binding to three dihydrofolate reductase mutants: (i) a mutant in which Asp-27, a residue that is directly involved in the binding of folates and antifolates but not NADPH, has been replaced by a serine, (ii) a mutant in which Phe-137 on the exterior of the molecule and distant from the binding sites has been replaced by a serine, and (iii) a mutant in which both Asp-27 and Phe-137 have been replaced by serines. Mutation of the Asp-27 residue reduces the affinity for NADPH by approximately 7-fold. Kinetic measurements have suggested that this is due mainly to an increase in the rate of dissociation of the initial complex and a slight shift in the enzyme equilibrium to favor the nonbinding conformation. The pH dependence of the conformer equilibrium is also shifted by approximately one pH unit to higher pH (pK approximately 6.5). In addition, the pH profile suggests the involvement of a second ionizable group having a pK of about 8 since, above pH 7, the proportion of the NADPH-binding form decreases.(ABSTRACT TRUNCATED AT 250 WORDS)
Dunn
S M
SM
Department of Pharmacology, University of Alberta, Edmonton, Canada.
Lanigan
T M
TM
Howell
E E
EE
D001224:Aspartic Acid; D001386:Azides / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003300:Copper / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D010649:Phenylalanine; D011485:Protein Binding; D011487:Protein Conformation; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism*
D001386:Azides; D001426:Bacterial Proteins; D001224:Aspartic Acid; D010649:Phenylalanine; D009249:NADP; D003300:Copper; D013762:Tetrahydrofolate Dehydrogenase