The kinetic mechanism of wild-type and mutant mouse dihydrofolate reductases.
A kinetic mechanism is presented for mouse dihydrofolate reductase that predicts all the steady-state parameters and full time-course kinetics. This mechanism was derived from association and dissociation rate constants and pre-steady-state transients by using stopped-flow fluorescence and absorbance measurements. The major features of this kinetic mechanism are as follows: (1) the two native enzyme conformers, E1 and E2, bind ligands with varying affinities although only one conformer, E1, can support catalysis in the forward direction, (2) tetrahydrofolate dissociation is the rate-limiting step under steady-state turnover at low pH, and (3) the pH-independent rate of hydride transfer from NADPH to dihydrofolate is fast (khyd = 9000 s-1) and favorable (Keq = 100). The overall mechanism is similar in form to the Escherichia coli kinetic scheme (Fierke et al., 1987), although several differences are observed: (1) substrates and products predominantly bind the same form of the E. coli enzyme, and (2) the hydride transfer rate from NADPH to either folate or dihydrofolate is considerably faster for the mouse enzyme. The role of Glu-30 (Asp-27 in E. coli) in mouse DHFR has also been examined by using site-directed mutagenesis as a potential source of these differences. While aspartic acid is strictly conserved in all bacterial DHFRs, glutamic acid is conserved in all known eucaryotes. The two major effects of substituting Asp for Glu-30 in the mouse enzyme are (1) a decreased rate of folate reduction and (2) an increased rate of hydride transfer from NADPH to dihydrofolate.(ABSTRACT TRUNCATED AT 250 WORDS)
Thillet
J
J
Institut Jacques Monod, Universite Paris VII, Place Jussieu, France.
Adams
J A
JA
Benkovic
S J
SJ
D000818:Animals; D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics; D005492:Folic Acid / Q000378:metabolism; D005971:Glutamates / Q000235:genetics; D018698:Glutamic Acid; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D051379:Mice; D009154:Mutation; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000378:metabolism
D005971:Glutamates; D001224:Aspartic Acid; D018698:Glutamic Acid; D005492:Folic Acid; D013762:Tetrahydrofolate Dehydrogenase