Coupling Interactions of Distal Residues Enhance Dihydrofolate Reductase Catalysis: Mutational Effects on Hydride Transfer Rates
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- 作者：P. T. Ravi Rajagopalan ; Stefan Lutz ; and Stephen J. Benkovic
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：October 22, 2002
- 年：2002
- 卷：41
- 期：42
- 页码：12618 - 12628
- 全文大小：165K
- 年卷期：v.41,no.42(October 22, 2002)
- ISSN：1520-4995
文摘
Recently, the participation in catalysis of residues spatially removed from the enzyme's activesite has received considerable attention. The influence of the distal Gly-121 residue on the chemical stepof hydride transfer in dihydrofolate reductase (DHFR) catalysis had been demonstrated previously[Cameron, C. E., and Benkovic, S. J. (1997) Biochemistry 36, 15792-15800]. In our continuing effort toidentify catalytically important residues that are distal from the active site, we used sequence conservationinformation, kinetic data on site-directed mutants, dynamic motion information from NMR methods, andcorrelated motions from MD simulations to identify a subset of residues. Among them, the region spanningpositions 41-45 is distal to the active site and was chosen as the focus for the mutagenesis and kineticstudies reported here. Specifically, the highly conserved Met-42 was selected for site-directed mutagenesis.While the reaction kinetics for the M42F mutant enzyme did not deviate from wild-type behavior, a41-fold reduction in the forward hydride transfer rate was found for the M42W mutant. Given the establishedrole of Gly-121 in the hydride transfer process, double mutant enzymes involving positions 42 and 121were constructed and characterized. These double mutant enzymes generally showed little changes insubstrate and cofactor binding but synergistic decreases in forward hydride transfer rates, while the decreasesin reverse rates were additive. Along with supporting information from mixed quantum/classical MDsimulations [Agarwal, P. K., Billeter, S. R., Rajagopalan, P. T., Benkovic, S. J., and Hammes-Schiffer,S. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 2794-2799], the data suggest that a coupled dynamic motionof these distal residues enhances crossing of the chemical reaction barrier and imply an expanded nonstaticrole for the protein fold in catalysis.