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Promiscuity in the Enzymatic Catalysis of Phosphate and Sulfate Transfer.

ABSTRACT: The enzymes that facilitate phosphate and sulfate hydrolysis are among the most proficient natural catalysts known to date. Interestingly, a large number of these enzymes are promiscuous catalysts that exhibit both phosphatase and sulfatase activities in the same active site and, on top of that, have also been demonstrated to efficiently catalyze the hydrolysis of other additional substrates with varying degrees of efficiency. Understanding the factors that underlie such multifunctionality is crucial both for understanding functional evolution in enzyme superfamilies and for the development of artificial enzymes. In this Current Topic, we have primarily focused on the structural and mechanistic basis for catalytic promiscuity among enzymes that facilitate both phosphoryl and sulfuryl transfer in the same active site, while comparing this to how catalytic promiscuity manifests in other promiscuous phosphatases. We have also drawn on the large number of experimental and computational studies of selected model systems in the literature to explore the different features driving the catalytic promiscuity of such enzymes. Finally, on the basis of this comparative analysis, we probe the plausible origins and determinants of catalytic promiscuity in enzymes that catalyze phosphoryl and sulfuryl transfer.

SUBMITTER: Pabis A

PROVIDER: S-EPMC4899807 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Promiscuity in the Enzymatic Catalysis of Phosphate and Sulfate Transfer.

Pabis Anna A Duarte Fernanda F Kamerlin Shina C L SC

Biochemistry 20160526 22

The enzymes that facilitate phosphate and sulfate hydrolysis are among the most proficient natural catalysts known to date. Interestingly, a large number of these enzymes are promiscuous catalysts that exhibit both phosphatase and sulfatase activities in the same active site and, on top of that, have also been demonstrated to efficiently catalyze the hydrolysis of other additional substrates with varying degrees of efficiency. Understanding the factors that underlie such multifunctionality is cr ...[more]

PMID: 27187273

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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.

Project description:[FeFe] hydrogenases, metalloenzymes catalyzing proton/dihydrogen interconversion, have attracted intense attention due to their remarkable catalytic properties and (bio-)technological potential for a future hydrogen economy. In order to unravel the factors enabling their efficient catalysis, both their unique organometallic cofactors and protein structural features, i.e., "outer-coordination sphere" effects have been intensively studied. These structurally diverse enzymes are divided into distinct phylogenetic groups, denoted as Group A-D. Prototypical Group A hydrogenases display high turnover rates (104-105 s-1). Conversely, the sole characterized Group D representative, Thermoanaerobacter mathranii HydS (TamHydS), shows relatively low catalytic activity (specific activity 10-1 μmol H2 mg-1 min-1) and has been proposed to serve a H2-sensory function. The various groups of [FeFe] hydrogenase share the same catalytic cofactor, the H-cluster, and the structural factors causing the diverging reactivities of Group A and D remain to be elucidated. In the case of the highly active Group A enzymes, a well-defined proton transfer pathway (PTP) has been identified, which shuttles H+ between the enzyme surface and the active site. In Group D hydrogenases, this conserved pathway is absent. Here, we report on the identification of highly conserved amino acid residues in Group D hydrogenases that constitute a possible alternative PTP. We varied two proposed key amino acid residues of this pathway (E252 and E289, TamHydS numbering) via site-directed mutagenesis and analyzed the resulting variants via biochemical and spectroscopic methods. All variants displayed significantly decreased H2-evolution and -oxidation activities. Additionally, the variants showed two redox states that were not characterized previously. These findings provide initial evidence that these amino acid residues are central to the putative PTP of Group D [FeFe] hydrogenase. Since the identified residues are highly conserved in Group D exclusively, our results support the notion that the PTP is not universal for different phylogenetic groups in [FeFe] hydrogenases.

Dataset Information

Promiscuity in the Enzymatic Catalysis of Phosphate and Sulfate Transfer.

Publications

Promiscuity in the Enzymatic Catalysis of Phosphate and Sulfate Transfer.

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