Monomeric metaphosphates

Westheimer, F. H.

doi:10.1021/cr00044a001

Cited by 208 publications

(118 citation statements)

References 22 publications

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“…Residues highlighted in red are highly conserved among the members of the novel ATP-binding family, residues highlighted in yellow are strongly conserved. Two possible mechanisms for amidine formation originally proposed by Westheimer (43). R = ribose-5′-phosphate, td = tetrahedral, ts = transition state.…”

Section: Resultsmentioning

confidence: 99%

Complexed Structures of Formylglycinamide Ribonucleotide Amidotransferase from Thermotoga maritima Describe a Novel ATP Binding Protein Superfamily^,

et al. 2006

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Formylglycinamide ribonucleotide amidotransferase (FGAR-AT) catalyzes the ATP-dependent synthesis of formylglycinamidine ribonucleotide (FGAM) from formylglycinamide ribonucleotide (FGAR) and glutamine in the fourth step of the purine biosynthetic pathway. FGAR-AT is encoded by the purL gene. Two types of PurL have been detected. The first type, found in eukaryotes and Gram-negative bacteria, consists of a single 140 kDa polypeptide chain and is designated large PurL (lgPurL). The second type, small PurL (smPurL), is found in archaea and Gram-positive bacteria and consists of an 80 kDa polypeptide chain. Small PurL requires two additional gene products, PurQ and PurS, for activity. PurL is a member of a protein superfamily that contains a novel ATP-binding domain. Structures of several members of this superfamily are available in the apo form. We determined five different structures of FGAR-AT from Thermotoga maritima in the presence of substrates, a substrate analog, and a product. These complexes have allowed a detailed description of the novel ATP-binding motif. Availability of a ternary complex enabled mapping of the active site thus identifying potential residues involved in catalysis. The complexes show a conformational change in the active site compared to the unliganded structure. A surprising discovery, an ATP molecule in an auxiliary site of the protein and the conformational changes associated with its binding, provoke speculations about the regulatory role of the auxiliary site in PurLSQ complex formation as well as the evolutionary relationship of PurL's from different organisms.The purine biosynthetic pathway is ubiquitous in most living organisms, and it is a ten-step process for converting phosphoribosyl pyrophosphate to inosine monophosphate. Formyl glycinamide ribonucleotide amidotransferase (FGAR-AT), also known by its gene product † This work was supported by the NIH grants RR15301 and GM073220 (SEE). SEE is indebted to the W. M. Keck Foundation and the Lucille P. Markey Charitable Trust. AAH was supported by a NSF predoctoral fellowship. AAH and JS were supported by NIH Grant GM32191. The Biophysical Instrumentation Facility for the Study of Complex Macromolecular Systems at MIT is supported by grants (NSF-0070319 and NIH GM68762). Supporting Information Available A table of primers used in TmPurL cloning and mutagenesis, a table summarizing the SV-AUC results, the Coomassie-stained gels for StPurL and mutants, a graph of the distribution of species obtained after SEDFIT analysis of SV-AUC data for wild type and mutant TmPurL, a topology diagram of TmPurL, circular dichroism spectra for wild type and mutant TmPurL, and an elution profile of wild type TmPurL after incubation with radioactive ATP. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 September 2. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript name, PurL, catalyzes the fourth...

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Section: Resultsmentioning

confidence: 99%

Complexed Structures of Formylglycinamide Ribonucleotide Amidotransferase from Thermotoga maritima Describe a Novel ATP Binding Protein Superfamily^,

et al. 2006

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“…2B). The electron density probably represents an equilibrium mixture of orthophosphate and PO 3 Ϫ /OH Ϫ . 97 , which in the control and high K ϩ complexes coordinates magnesium cations at sites 2 and 3, now bridges the magnesium cations at sites 1 and 2.…”

Section: Resultsmentioning

confidence: 99%

“…The transfer of the phosphoryl group of a phosphate ester to water in most cases requires metal ions as cofactors, and proceeds either by way of a trigonal-bipyramidal transition state (associative mechanism), or through the formation of an unstable intermediate of metaphosphate (dissociative mechanism) (1Ϫ6). The metaphosphate anion (PO 3 Ϫ ) was first proposed as an intermediate in the hydrolysis of phosphate esters nearly 50 years ago (7,8). Although it exists as a stable entity in the gas phase, where it is relatively non-reactive (9), metaphosphate is unstable in aqueous solutions, and its existence is inferred only by indirect evidence (9Ϫ15).…”

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confidence: 99%

Metaphosphate in the Active Site of Fructose-1,6-bisphosphatase

Choe

Iancu

Fromm

et al. 2003

Journal of Biological Chemistry

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The hydrolysis of a phosphate ester can proceed through an intermediate of metaphosphate (dissociative mechanism) or through a trigonal bipryamidal transition state (associative mechanism). Model systems in solution support the dissociative pathway, whereas most enzymologists favor an associative mechanism for enzyme-catalyzed reactions. Crystals of fructose-1,6-bisphosphatase grow from an equilibrium mixture of substrates and products at near atomic resolution (1.3 Å). At neutral pH, products of the reaction (orthophosphate and fructose 6-phosphate) bind to the active site in a manner consistent with an associative reaction pathway; however, in the presence of inhibitory concentrations of K ؉ (200 mM), or at pH 9.6, metaphosphate and water (or OH ؊ ) are in equilibrium with orthophosphate. Furthermore, one of the magnesium cations in the pH 9.6 complex resides in an alternative position, and suggests the possibility of metal cation migration as the 1-phosphoryl group of the substrate undergoes hydrolysis. To the best of our knowledge, the crystal structures reported here represent the first direct observation of metaphosphate in a condensed phase and may provide the structural basis for fundamental changes in the catalytic mechanism of fructose-1,6-bisphosphatase in response to pH and different metal cation activators.Phosphatases, mutases, kinases, and nucleases all catalyze phosphoryl transfer reactions central to biochemical processes that sustain life. The transfer of the phosphoryl group of a phosphate ester to water in most cases requires metal ions as cofactors, and proceeds either by way of a trigonal-bipyramidal transition state (associative mechanism), or through the formation of an unstable intermediate of metaphosphate (dissociative mechanism) (1Ϫ6). The metaphosphate anion (PO 3 Ϫ ) was first proposed as an intermediate in the hydrolysis of phosphate esters nearly 50 years ago (7,8). Although it exists as a stable entity in the gas phase, where it is relatively non-reactive (9), metaphosphate is unstable in aqueous solutions, and its existence is inferred only by indirect evidence (9Ϫ15). The likelihood of trapping metaphosphate in the active site of an enzyme is remote, because of the proximity of acceptor molecules in the active site. Yet an enzyme offers an advantage in that it reduces the free energy of the transition state. Hence, the active site itself could serve as a thermodynamic trap if metaphosphate, once generated, is denied access to an acceptor.Fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11, hereafter FBPase) 1 is a key regulatory enzyme in gluconeogenesis. FBPase catalyzes the hydrolysis of fructose 1,6-bisphosphate (F16P 2 ) to fructose 6-phosphate (F6P) and orthophosphate (P i ). The inhibition of FBPase in mammals results in reduced levels of serum glucose in the fasting state. Hence, FBPase is a target for the development of drugs in the treatment of non-insulin dependent diabetes, which afflicts over 15 million people in the United State...

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“…Phosphate hydrolysis reactions are arguably the most important class of biological reactions (7)(8)(9)(10)(11), and it is thus important to elucidate the relevant mechanism in solution and proteins. In this respect, it is now gradually realized that QM(ai)/MM studies are perhaps the only way of resolving the fundamental mechanistic controversies associated with phosphate hydrolysis (11).…”

Section: Quantifying the Relationship Between Competing Mechanisms Ofmentioning

confidence: 99%

Quantitative exploration of the molecular origin of the activation of GTPase

Plotnikov

Lameira

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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GTPases play a major role in cellular processes, and gaining quantitative understanding of their activation demands reliable free energy surfaces of the relevant mechanistic paths in solution, as well as the interpolation of this information to GTPases. Recently, we generated ab initio quantum mechanical/molecular mechanical free energy surfaces for the hydrolysis of phosphate monoesters in solution, establishing quantitatively that the barrier for the reactions with a proton transfer (PT) step from a single attacking water (1W) is higher than the one where the PT is assisted by a second water (2W). The implication of this finding on the activation of GTPases is quantified here, by using the ab initio solution surfaces to calibrate empirical valence bond surfaces and then exploring the origin of the activation effect. It is found that, although the 2W PT path is a new element, this step is not rate determining, and the catalytic effect is actually due to the electrostatic stabilization of the pre-PT transition state and the subsequent plateau. Thus, the electrostatic catalytic effect found in our previous studies of the Ras GTPase activating protein (RasGAP) and the elongation factor-Tu (EF-Tu) with a 1W mechanism is still valid for the 2W path. Furthermore, as found before, the corresponding activation appears to involve a major allosteric effect. Overall, we believe that our finding is general to both GTPases and ATPases. In addition to the biologically relevant finding, we also provide a critical discussion of the requirements from reliable surfaces for enzymatic reactions.D etailed understanding of many problems in biology boils down to the elucidation of the correct reaction mechanism in the protein and in solution and to the elucidation of the origin of the corresponding catalytic effect. However, this requires overcoming the challenge of obtaining accurate free energy surfaces in the condensed phase. Such a task can be accomplished, in principle, by the combined quantum mechanical/molecular mechanical (QM/ MM) method (1-5). However, the implementation of this method is still very challenging. At present, we are not at the stage reached in studies of gas phase reactions, where the results of ab initio calculations are sometimes considered almost as substitutes for experimental results. Nevertheless, recent advances in ab initio QM/MM [QM(ai)/MM] studies (2, 4-6) brought us closer to having quantitative results for solution reactions and in some respects to quantitative results for enzymes, especially if one evaluates reliable QM (ai)/MM free energy surfaces for the solution reaction and then use the resulting surfaces to calibrate empirical valence bond (EVB) surfaces for studying the relevant enzymatic reaction. Here we exploit these advances by exploring the hydrolysis of phosphate monoesters that is the key to the activation of GTPases. That is, we start by quantifying the energetics of the solution reaction and then use the solution free energy surface to calibrate EVB surface that allow us to reliably...

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Monomeric metaphosphates

Cited by 208 publications

References 22 publications

Complexed Structures of Formylglycinamide Ribonucleotide Amidotransferase from Thermotoga maritima Describe a Novel ATP Binding Protein Superfamily^,

Complexed Structures of Formylglycinamide Ribonucleotide Amidotransferase from Thermotoga maritima Describe a Novel ATP Binding Protein Superfamily^,

Metaphosphate in the Active Site of Fructose-1,6-bisphosphatase

Quantitative exploration of the molecular origin of the activation of GTPase

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Monomeric metaphosphates

Cited by 208 publications

References 22 publications

Complexed Structures of Formylglycinamide Ribonucleotide Amidotransferase from Thermotoga maritima Describe a Novel ATP Binding Protein Superfamily,

Complexed Structures of Formylglycinamide Ribonucleotide Amidotransferase from Thermotoga maritima Describe a Novel ATP Binding Protein Superfamily,

Metaphosphate in the Active Site of Fructose-1,6-bisphosphatase

Quantitative exploration of the molecular origin of the activation of GTPase

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Product

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Complexed Structures of Formylglycinamide Ribonucleotide Amidotransferase from Thermotoga maritima Describe a Novel ATP Binding Protein Superfamily^,

Complexed Structures of Formylglycinamide Ribonucleotide Amidotransferase from Thermotoga maritima Describe a Novel ATP Binding Protein Superfamily^,