Research Article

The role of dimer asymmetry and protomer dynamics in enzyme catalysis

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Science  20 Jan 2017:
Vol. 355, Issue 6322, eaag2355
DOI: 10.1126/science.aag2355

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Working as a pair

Enzymes provide scaffolds that facilitate chemical reactions. Enzyme dynamics often enhance reactivity by allowing the enzyme to sample the transition state between reactants and products. Kim et al. explored the role of dynamics in the dimeric enzyme fluoroacetate dehalogenase (see the Perspective by Saleh and Kalodimos). They found that the two protomers are asymmetric, with only one being able to bind substrate at a time. The nonbinding protomer contributed to catalysis by becoming more dynamic to compensate for the entropy loss of its partner.

Science, this issue p. 10.1126/science.aag2355 ; see also p. 247

Structured Abstract

INTRODUCTION

Enzymes greatly accelerate biochemical reactions by providing a scaffold to bind and recognize substrate, position catalytic units, and facilitate the formation of stabilized transition states. The conformations associated with specific states along the reaction coordinate pathway are often observed to be sampled by the enzyme through a conformational selection mechanism. An enzyme-catalyzed reaction is not a simple linear process of discrete steps where the protein progresses in an ordered and sequential fashion. Rather, a more appropriate description is in terms of an ensemble of functional conformations. Here, we consider how such ensembles might function in the homodimeric enzyme fluoroacetate dehalogenase (FAcD) from Rhodopseudomonas palustris. Even under gross excess of substrate, this enzyme turns over only one substrate molecule at a time, raising the question of the role played by the dimer in catalysis.

Our results indicate that substrate binding to one protomer is allosterically communicated to the empty protomer, which plays a critical role in facilitating catalysis through entropic changes manifesting in enhanced dynamics and the loss of bound water molecules. Several key mutants were prepared so that the enzyme could also be studied in various stages associated with catalysis (i.e. substrate-free, Michaelis intermediate, covalent intermediate, and product-bound state). By characterizing these functional states using x-ray crystallography and solution-state nuclear magnetic resonance (NMR), it became possible to invoke an ensemble description of the enzyme at key points along the reaction. Interconversion between conformers and the dynamic allosteric processes associated with driving catalysis in the enzyme could be studied largely through 19F NMR experiments, whereas the dynamic ensemble and key allosteric processes were validated by molecular dynamics (MD) simulations and computational rigidity analysis.

RATIONALE

Catalytic turnover numbers for most enzymes are between 1 and 104 s–1. FAcD turnover, however, is conveniently slow (several substrate molecules per minute), making it feasible to interrogate functional states associated with unidirectional catalysis by means of freeze-trapping x-ray crystallography and NMR. Our goal was to investigate key functional mutants in order to derive an ensemble representation of the enzyme and to better understand how this ensemble achieves catalysis. The work provides new insights into the role of allostery and protein dynamics in catalysis, and in particular, how this takes place in a dimeric enzyme.

RESULTS

Crystallographic analysis identifies a subtle asymmetry in the dimer, where only one protomer is poised for substrate binding at any instance. NMR reveals that the two protomers undergo conformational exchange on a millisecond time scale. In the absence of substrate, however, 0.5% of the enzyme molecules adopt an asymmetric ligand bound-like excited state with little or no conformational exchange. Upon binding of substrate, the asymmetry becomes more pronounced, and the empty protomer contributes to catalysis by shedding water molecules and adopting greater short–time scale fluctuations, thereby compensating for entropy losses associated with binding. In addition, conformational exchange between protomers markedly increases once the substrate is locked into the binding cleft. Substrate binding also initiates sampling of the covalent intermediate, and subsequent functional states on a millisecond time scale. Water networks appear to be a hallmark of key functional states and to play a role in allostery. MD simulations and rigidity theory effectively identify intramolecular and interprotomer allosteric pathways, which drive catalysis.

CONCLUSION

FAcD effectively reacts one substrate at a time. However, the empty half of the dimer plays a key role in sampling subsequent functional states and compensating for entropy penalties to binding. Indeed, subsequent states in the catalytic process appear to be sampled through a conformational selection mechanism, in which the empty protomer plays a key role. MD simulations and rigidity theory help to validate many key ideas associated with sampling of states within the ensemble, allostery, and the role of protein dynamics in catalysis.

Heterogeneous dynamics and structural waters in FAcD.

In the apostate, FAcD has a comparable number of waters and B-factor amplitudes in each protomer. Binding of substrate increases B factors and causes an egress of water molecules in the empty protomer. Upon SN2 substitution, there is a decrease in B factors and an influx of water molecules that continues upon hydrolysis and product formation.

Abstract

Freeze-trapping x-ray crystallography, nuclear magnetic resonance, and computational techniques reveal the distribution of states and their interconversion rates along the reaction pathway of a bacterial homodimeric enzyme, fluoroacetate dehalogenase (FAcD). The crystal structure of apo-FAcD exhibits asymmetry around the dimer interface and cap domain, priming one protomer for substrate binding. This asymmetry is dynamically averaged through conformational exchange on a millisecond time scale. During catalysis, the protomer conformational exchange rate becomes enhanced, the empty protomer exhibits increased local disorder, and water egresses. Computational studies identify allosteric pathways between protomers. Water release and enhanced dynamics associated with catalysis compensate for entropic losses from substrate binding while facilitating sampling of the transition state. The studies provide insights into how substrate-coupled allosteric modulation of structure and dynamics facilitates catalysis in a homodimeric enzyme.

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