Dataset Information

Protein Flexibility and Stiffness Enable Efficient Enzymatic Catalysis.

ABSTRACT: The enormous rate accelerations observed for many enzyme catalysts are due to strong stabilizing interactions between the protein and reaction transition state. The defining property of these catalysts is their specificity for binding the transition state with a much higher affinity than substrate. Experimental results are presented which show that the phosphodianion-binding energy of phosphate monoester substrates is used to drive conversion of their protein catalysts from flexible and entropically rich ground states to stiff and catalytically active Michaelis complexes. These results are generalized to other enzyme-catalyzed reactions. The existence of many enzymes in flexible, entropically rich, and inactive ground states provides a mechanism for utilization of ligand-binding energy to mold these catalysts into stiff and active forms. This reduces the substrate-binding energy expressed at the Michaelis complex, while enabling the full and specific expression of large transition-state binding energies. Evidence is presented that the complexity of enzyme conformational changes increases with increases in the enzymatic rate acceleration. The requirement that a large fraction of the total substrate-binding energy be utilized to drive conformational changes of floppy enzymes is proposed to favor the selection and evolution of protein folds with multiple flexible unstructured loops, such as the TIM-barrel fold. The effect of protein motions on the kinetic parameters for enzymes that undergo ligand-driven conformational changes is considered. The results of computational studies to model the complex ligand-driven conformational change in catalysis by triosephosphate isomerase are presented.

SUBMITTER: Richard JP

PROVIDER: S-EPMC6396832 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Protein Flexibility and Stiffness Enable Efficient Enzymatic Catalysis.

Richard John P JP

Journal of the American Chemical Society 20190214 8

The enormous rate accelerations observed for many enzyme catalysts are due to strong stabilizing interactions between the protein and reaction transition state. The defining property of these catalysts is their specificity for binding the transition state with a much higher affinity than substrate. Experimental results are presented which show that the phosphodianion-binding energy of phosphate monoester substrates is used to drive conversion of their protein catalysts from flexible and entropic ...[more]

PMID: 30703322

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Project description:CONSPECTUS: As is well-known, enzymes are proteins designed to accelerate specific life essential chemical reactions by many orders of magnitude. A folded protein is a highly dynamical entity, best described as a hierarchy or ensemble of interconverting conformations on all time scales from femtoseconds to minutes. We are just beginning to learn what role these dynamics play in the mechanism of chemical catalysis by enzymes due to extraordinary difficulties in characterizing the conformational space, that is, the energy landscape, of a folded protein. It seems clear now that their role is crucially important. Here we discuss approaches, based on vibrational spectroscopies of various sorts, that can reveal the energy landscape of an enzyme-substrate (Michaelis) complex and decipher which part of the typically very complicated landscape is relevant to catalysis. Vibrational spectroscopy is quite sensitive to small changes in bond order and bond length, with a resolution of 0.01 Å or less. It is this sensitivity that is crucial to its ability to discern bond reactivity. Using isotope edited IR approaches, we have studied in detail the role of conformational heterogeneity and dynamics in the catalysis of hydride transfer by LDH (lactate dehydrogenase). Upon the binding of substrate, the LDH·substrate system undergoes a search through conformational space to find a range of reactive conformations over the microsecond to millisecond time scale. The ligand is shuttled to the active site via first forming a weakly bound enzyme·ligand complex, probably consisting of several heterogeneous structures. This complex undergoes numerous conformational changes spread throughout the protein that shuttle the enzyme·substrate complex to a range of conformations where the substrate is tightly bound. This ensemble of conformations all have a propensity toward chemistry, but some are much more facile for carrying out chemistry than others. The search for these tightly bound states is clearly directed by the forces that the protein can bring to bear, very much akin to the folding process to form native protein in the first place. In fact, the conformational subspace of reactive conformations of the Michaelis complex can be described as a "collapse" of reactive substates compared with that found in solution, toward a much smaller and much more reactive set. These studies reveal how dynamic disorder in the protein structure can modulate the on-enzyme reactivity. It is very difficult to account for how the dynamical nature of the ground state of the Michaelis complex modulates function by transition state concepts since dynamical disorder is not a starting feature of the theory. We find that dynamical disorder may well play a larger or similar sized role in the measured Gibbs free energy of a reaction compared with the actual energy barrier involved in the chemical event. Our findings are broadly compatible with qualitative concepts of evolutionary adaptation of function such as adaptation to varying thermal environments. Our work suggests a methodology to determine the important dynamics of the Michaelis complex.

Dataset Information

Protein Flexibility and Stiffness Enable Efficient Enzymatic Catalysis.

Publications

Protein Flexibility and Stiffness Enable Efficient Enzymatic Catalysis.

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