Effects of temperature and viscosity on R67 dihydrofolate reductase catalysis.

Abstract:

:R67 dihydrofolate reductase (DHFR) is a novel homotetrameric protein that possesses 222 symmetry and a single, voluminous active site pore. This symmetry poses numerous limitations on catalysis; for example, two dihydrofolate (DHF) molecules or two NADPH molecules, or one substrate plus one cofactor can bind. Only the latter combination leads to catalysis. To garner additional information on how this enzyme facilitates transition-state formation, the temperature dependence of binding and catalysis was monitored. The binding of NADPH and DHF is enthalpy-driven. Previous primary isotope effect studies indicate hydride transfer is at least partially rate-determining. Accordingly, the activation energy associated with transition-state formation was measured and is found to be 6.9 kcal/mol (DeltaH(++)(25) = 6.3 kcal/mol). A large entropic component is also found associated with catalysis, TDeltaS(++)(25) = -11.3 kcal/mol. The poor substrate, dihydropteroate, binds more weakly than dihydrofolate (DeltaDeltaG = 1.4 kcal/mol) and displays a large loss in the binding enthalpy value (DeltaDeltaH = 3.8 kcal/mol). The k(cat) value for dihydropteroate reduction is decreased 1600-fold compared to DHF usage. This effect appears to derive mostly from the DeltaDeltaH difference in binding, demonstrating that the glutamate tail is important for catalysis. This result is surprising, as the para-aminobenzoyl-glutamate tail of DHF has been previously shown to be disordered by both NMR and crystallography studies. Viscosity studies were also performed and confirmed that the hydride transfer rate is not sensitive to sucrose addition. Surprisingly, binding of DHF, by both K(m) and K(d) determination, was found to be sensitive to added viscogens, suggesting a role for water in DHF binding.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Chopra S,Lynch R,Kim SH,Jackson M,Howell EE

doi

10.1021/bi052504l

subject

Has Abstract

pub_date

2006-05-30 00:00:00

pages

6596-605

issue

21

eissn

0006-2960

issn

1520-4995

journal_volume

45

pub_type

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