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Multiple intermediates, diverse conformations, and cooperative conformational changes underlie the catalytic hydride transfer reaction of dihydrofolate reductase.

ABSTRACT: It has become increasingly clear that protein motions play an essential role in enzyme catalysis. However, exactly how these motions are related to an enzyme's chemical step is still intensely debated. This chapter examines the possible role of protein motions that display a hierarchy of timescales in enzyme catalysis. The linkage between protein motions and catalysis is investigated in the context of a model enzyme, E. coli dihydrofolate reductase (DHFR), that catalyzes the hydride transfer reaction in the conversion of dihydrofolate to tetrahydrofolate. The results of extensive computer simulations probing the protein motions that are manifest during different steps along the turnover cycle of DHFR are summarized. Evidence is presented that the protein motions modulate the catalytic efficacy of DHFR by generating a conformational ensemble conducive to the hydride transfer. The alteration of the equilibrium conformational ensemble rather than any protein dynamical effects is found to be sufficient to explain the rate-diminishing effects of mutation on the kinetics of the enzyme. These data support the view that the protein motions facilitate catalysis by establishing reaction competent conformations of the enzyme, but they do not directly couple to the chemical reaction itself. These findings have broad implications for our understanding of enzyme mechanisms and the design of novel protein catalysts.

SUBMITTER: Arora K

PROVIDER: S-EPMC4394636 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Multiple intermediates, diverse conformations, and cooperative conformational changes underlie the catalytic hydride transfer reaction of dihydrofolate reductase.

Arora Karunesh K Brooks Charles L CL

Topics in current chemistry 20130101

It has become increasingly clear that protein motions play an essential role in enzyme catalysis. However, exactly how these motions are related to an enzyme's chemical step is still intensely debated. This chapter examines the possible role of protein motions that display a hierarchy of timescales in enzyme catalysis. The linkage between protein motions and catalysis is investigated in the context of a model enzyme, E. coli dihydrofolate reductase (DHFR), that catalyzes the hydride transfer rea ...[more]

PMID: 23420416

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Project description:The three-dimensional structures of the dihydrofolate reductase enzymes from Escherichia coli (ecDHFR or ecE) and Homo sapiens (hDHFR or hE) are very similar, despite a rather low level of sequence identity. Whereas the active site loops of ecDHFR undergo major conformational rearrangements during progression through the reaction cycle, hDHFR remains fixed in a closed loop conformation in all of its catalytic intermediates. To elucidate the structural and dynamic differences between the human and E. coli enzymes, we conducted a comprehensive analysis of side chain flexibility and dynamics in complexes of hDHFR that represent intermediates in the major catalytic cycle. Nuclear magnetic resonance relaxation dispersion experiments show that, in marked contrast to the functionally important motions that feature prominently in the catalytic intermediates of ecDHFR, millisecond time scale fluctuations cannot be detected for hDHFR side chains. Ligand flux in hDHFR is thought to be mediated by conformational changes between a hinge-open state when the substrate/product-binding pocket is vacant and a hinge-closed state when this pocket is occupied. Comparison of X-ray structures of hinge-open and hinge-closed states shows that helix αF changes position by sliding between the two states. Analysis of χ1 rotamer populations derived from measurements of (3)JCγCO and (3)JCγN couplings indicates that many of the side chains that contact helix αF exhibit rotamer averaging that may facilitate the conformational change. The χ1 rotamer adopted by the Phe31 side chain depends upon whether the active site contains the substrate or product. In the holoenzyme (the binary complex of hDHFR with reduced nicotinamide adenine dinucleotide phosphate), a combination of hinge opening and a change in the Phe31 χ1 rotamer opens the active site to facilitate entry of the substrate. Overall, the data suggest that, unlike ecDHFR, hDHFR requires minimal backbone conformational rearrangement as it proceeds through its enzymatic cycle, but that ligand flux is brokered by more subtle conformational changes that depend on the side chain motions of critical residues.

Dataset Information

Multiple intermediates, diverse conformations, and cooperative conformational changes underlie the catalytic hydride transfer reaction of dihydrofolate reductase.

Publications

Multiple intermediates, diverse conformations, and cooperative conformational changes underlie the catalytic hydride transfer reaction of dihydrofolate reductase.

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