Kinetic investigation of the functional role of phenylalanine-31 of recombinant human dihydrofolate reductase.
The role of the active site residue phenylalanine-31 (Phe31) for recombinant human dihydrofolate reductase (rHDHFR) has been probed by comparing the kinetic behavior of wild-type enzyme (wt) with mutant in which Phe31 is replaced by leucine (F31L rHDHFR). At pH 7.65 the steady-state kcat is almost doubled, but the rate constant for hydride transfer is decreased to less than half that for wt enzyme, as is the rate of the obligatory isomerization of the substrate complex that precedes hydride transfer. Although steady-state measurements indicated that the mutation causes large increases in Km for both substrates, dissociation constants for many complexes are decreased. These apparent paradoxes are due to major mutation-induced decreases in rate constants (koff) for dissociation of folate, dihydrofolate, and tetrahydrofolate from all of their complexes. This results in a mechanism proceeding almost entirely by only one of the two pathways used by wt enzyme. Other consequences of these changes are a much altered dependence of steady-state kcat on pH, inhibition rather than activation by tetrahydrofolate, absence of hysteresis in transient-state kinetics, and a decrease in enzyme efficiency under physiological conditions. The results indicate that there is no quantitative correlation between dihydrofolate binding and the rate of hydride transfer for this enzyme.
Tsay
J T
JT
Department of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101.
Appleman
J R
JR
Beard
W A
WA
Prendergast
N J
NJ
Delcamp
T J
TJ
Freisheim
J H
JH
Blakley
R L
RL
D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites; D001708:Biopterins / Q000031:analogs & derivatives / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007930:Leucine; D010649:Phenylalanine / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism*; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*; D013763:Tetrahydrofolates / Q000378:metabolism
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D013763:Tetrahydrofolates; D001708:Biopterins; D010649:Phenylalanine; C017226:7,8-dihydrobiopterin; D013762:Tetrahydrofolate Dehydrogenase; D007930:Leucine