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Correlated structural kinetics and retarded solvent dynamics at the metalloprotease active site.

ABSTRACT: Solvent dynamics can play a major role in enzyme activity, but obtaining an accurate, quantitative picture of solvent activity during catalysis is quite challenging. Here, we combine terahertz spectroscopy and X-ray absorption analyses to measure changes in the coupled water-protein motions during peptide hydrolysis by a zinc-dependent human metalloprotease. These changes were tightly correlated with rearrangements at the active site during the formation of productive enzyme-substrate intermediates and were different from those in an enzyme-inhibitor complex. Molecular dynamics simulations showed a steep gradient of fast-to-slow coupled protein-water motions around the protein, active site and substrate. Our results show that water retardation occurs before formation of the functional Michaelis complex. We propose that the observed gradient of coupled protein-water motions may assist enzyme-substrate interactions through water-polarizing mechanisms that are remotely mediated by the catalytic metal ion and the enzyme active site.

SUBMITTER: Grossman M

PROVIDER: S-EPMC3308126 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Correlated structural kinetics and retarded solvent dynamics at the metalloprotease active site.

Grossman Moran M Born Benjamin B Heyden Matthias M Tworowski Dmitry D Fields Gregg B GB Sagi Irit I Havenith Martina M

Nature structural & molecular biology 20110918 10

Solvent dynamics can play a major role in enzyme activity, but obtaining an accurate, quantitative picture of solvent activity during catalysis is quite challenging. Here, we combine terahertz spectroscopy and X-ray absorption analyses to measure changes in the coupled water-protein motions during peptide hydrolysis by a zinc-dependent human metalloprotease. These changes were tightly correlated with rearrangements at the active site during the formation of productive enzyme-substrate intermedia ...[more]

PMID: 21926991

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Project description:In two-component signal transduction, response regulator proteins contain the catalytic machinery for their own covalent phosphorylation and can catalyze phosphotransfer from a partner sensor kinase or autophosphorylate using various small molecule phosphodonors. Although response regulator autophosphorylation is physiologically relevant and a powerful experimental tool, the kinetic determinants of the autophosphorylation reaction and how those determinants might vary for different response regulators and phosphodonors are largely unknown. We characterized the autophosphorylation kinetics of 21 variants of the model response regulator Escherichia coli CheY that contained substitutions primarily at nonconserved active site positions D + 2 (CheY residue 59) and T + 2 (CheY residue 89), two residues C-terminal to conserved D57 and T87, respectively. Overall, the CheY variants exhibited a >10(5)-fold range of rate constants (kphos/KS) for reaction with phosphoramidate, acetyl phosphate, or monophosphoimidazole, with the great majority of rates enhanced versus that of wild-type CheY. Although phosphodonor preference varied substantially, nearly all the CheY variants reacted faster with phosphoramidate than acetyl phosphate. Correlation between the increased positive charge of the D + 2 and T + 2 side chains and faster rates indicated electrostatic interactions are a kinetic determinant. Moreover, sensitivities of rate constants to ionic strength indicated that both long-range and localized electrostatic interactions influence autophosphorylation kinetics. The increased nonpolar surface area of the D + 2 and T + 2 side chains also correlated with an enhanced autophosphorylation rate, especially for reaction with phosphoramidate and monophosphoimidazole. Computer docking suggested that highly accelerated monophosphoimidazole autophosphorylation rates for CheY variants with a tyrosine at position T + 2 likely reflect structural mimicry of phosphotransfer from the sensor kinase histidyl phosphate.

Dataset Information

Correlated structural kinetics and retarded solvent dynamics at the metalloprotease active site.

Publications

Correlated structural kinetics and retarded solvent dynamics at the metalloprotease active site.

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