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Effects of a distal mutation on active site chemistry.

Abstract：

:Previous studies of Escherichia coli dihydrofolate reductase (ecDHFR) have demonstrated that residue G121, which is 19 A from the catalytic center, is involved in catalysis, and long distance dynamical motions were implied. Specifically, the ecDHFR mutant G121V has been extensively studied by various experimental and theoretical tools, and the mutation's effect on kinetic, structural, and dynamical features of the enzyme has been explored. This work examined the effect of this mutation on the physical nature of the catalyzed hydride transfer step by means of intrinsic kinetic isotope effects (KIEs), their temperature dependence, and activation parameters as described previously for wild type ecDHFR [Sikorski, R. S., et al. (2004) J. Am. Chem. Soc. 126, 4778-4779]. The temperature dependence of initial velocities was used to estimate activation parameters. Isotope effects on the preexponential Arrhenius factors, and the activation energy, could be rationalized by an environmentally coupled hydrogen tunneling model, similar to the one used for the wild-type enzyme. Yet, in contrast to that in the wild type, fluctuations of the donor-acceptor distance were now required. Secondary (2 degrees ) KIEs were also measured for both H- and D-transfer, and as in the case of the wild-type enzyme, no coupled motion was detected. Despite these similarities, the reduced rates, the slightly inflated primary (1 degrees ) KIEs, and their temperature dependence, together with relatively deflated 2 degrees KIEs, indicate that the potential surface prearrangement was not as ideal as for the wild-type enzyme. These findings support theoretical studies suggesting that the G121V mutation led to a different conformational ensemble of reactive states and less effective rearrangement of the potential surface but has an only weak effect on H-tunneling.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Wang L,Tharp S,Selzer T,Benkovic SJ,Kohen A

doi

10.1021/bi0518242

subject

Has Abstract

pub_date

2006-02-07 00:00:00

pages

1383-92

issue

5

eissn

0006-2960

issn

1520-4995

journal_volume

45

pub_type

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