Role of the conserved active site residue tryptophan-24 of human dihydrofolate reductase as revealed by mutagenesis.
The active sites of all bacterial and vertebrate dihydrofolate reductases that have been examined have a tryptophan residue near the binding sites for NADPH and dihydrofolate. In cases where the three-dimensional structure has been determined by X-ray crystallography, this conserved tryptophan residue makes hydrophobic and van der Waals interactions with the nicotinamide moiety of bound NADPH, and its indole nitrogen interacts with the C4 oxygen of bound folate through a bridge provided by a bound water molecule. We have addressed the question of why even the very conservative replacement of this tryptophan by phenylalanine does not seem to occur naturally. Human dihydrofolate reductase with this replacement of tryptophan by phenylalanine has increased rate constants for dissociation of substrates and products and a considerably decreased rate of hydride transfer. These cause some changes in steady-state kinetic behavior, including substantial increases in Michaelis constants for NADPH and dihydrofolate, but there is also a 3-fold increase in kcat. The branched mechanistic pathway for this enzyme has been completely defined and is sufficiently different from that of wild-type enzyme to cause changes in some transient-state kinetics. The most critical changes resulting from the amino acid substitution appear to be a 50% decrease in stability and a decrease in efficiency from 69% to 21% under intracellular conditions.
Beard
W A
WA
Department of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101.
Appleman
J R
JR
Huang
S M
SM
Delcamp
T J
TJ
Freisheim
J H
JH
Blakley
R L
RL
D000494:Allosteric Regulation; D001051:Apoenzymes / Q000378:metabolism; D001665:Binding Sites; D006801:Humans; D006859:Hydrogen / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D009154:Mutation; D009249:NADP / Q000378:metabolism; D010649:Phenylalanine / Q000502:physiology; D011489:Protein Denaturation; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D013763:Tetrahydrofolates / Q000378:metabolism; D014364:Tryptophan / Q000502:physiology
D001051:Apoenzymes; D008024:Ligands; D013763:Tetrahydrofolates; D010649:Phenylalanine; D009249:NADP; D006859:Hydrogen; D014364:Tryptophan; D013762:Tetrahydrofolate Dehydrogenase