Chemical Confirmation of a Pentavalent Phosphorane in Complex with β-Phosphoglucomutase

Tremblay, L.W.; Zhang, Guofeng; Dai, Jianying; Dunaway‐Mariano, Debra; Allen, Karen N.

doi:10.1021/ja0509073

Cited by 57 publications

(66 citation statements)

References 8 publications

(14 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The electron density map is also consistent with a TS analogue (TSA) formed from a five-coordinate magnesium surrounded by two oxygen and three fluoride ligands (MgF 3 -TSA) that mimics the phosphoryl group in flight (12,13). The crystallization buffer needs to contain magnesium and fluoride ions for crystals to form (14,15), so all of the components necessary for the formation of the MgF 3 -TSA are available. Furthermore, ab initio quantummechanical calculations indicate that MgF 3 -TSA is stable, whereas INT is not and spontaneously rearranges to a phosphate monoester covalently bound to either the substrate or the enzyme, as would be expected of a transition state (16).…”

supporting

confidence: 52%

“…5). However, the published inhibition study was conducted under the unusual conditions of a 2-fold excess of intermediate ␤-G16BP (200 M) to substrate ␤-G1P (100 M) (15). When we repeated our experiments in the presence of high concentrations of ␤-G16BP, we also observed that even 10 mM fluoride did not inhibit the reaction (Fig.…”

Section: Resultsmentioning

confidence: 74%

“…For the remainder of ␤-PGM, small ⌬␦ indicate that, on formation of MgF3-TSA, there is little change in the local protein fold outside of these regions. that kinetic assays of ␣-and ␤-PGM are usually run in the presence of either ␣-or ␤-G16BP, which accelerate the reaction by saturating the enzyme at the intermediate stage or maintaining its activated, phosphorylated state (10,15,19). Only ␤-G16BP can contribute by maintaining a high concentration of the ␤-G16BP͞␤-PGM complex, but other cofactors can stimulate the enzyme by acting as phosphorylating agents, and commercially available ␣-G16BP is used commonly (10,(23)(24)(25).…”

Section: Resultsmentioning

confidence: 99%

See 2 more Smart Citations

A Trojan horse transition state analogue generated by MgF ₃ ⁻ formation in an enzyme active site

Baxter

Olguín

Goličnik

et al. 2006

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

113

View full text Add to dashboard Cite

Identifying how enzymes stabilize high-energy species along the reaction pathway is central to explaining their enormous rate acceleration. ␤-Phosphoglucomutase catalyses the isomerization of ␤-glucose-1-phosphate to ␤-glucose-6-phosphate and appeared to be unique in its ability to stabilize a high-energy pentacoordinate phosphorane intermediate sufficiently to be directly observable in the enzyme active site. Using 19 F-NMR and kinetic analysis, we report that the complex that forms is not the postulated high-energy reaction intermediate, but a deceptively similar transition state analogue in which MgF 3 ؊ mimics the transferring PO 3 ؊ moiety. Here we present a detailed characterization of the metal ion-fluoride complex bound to the enzyme active site in solution, which reveals the molecular mechanism for fluoride inhibition of ␤-phosphoglucomutase. This NMR methodology has a general application in identifying specific interactions between fluoride complexes and proteins and resolving structural assignments that are indistinguishable by x-ray crystallography.enzyme mechanism ͉ fluoride inhibition ͉ NMR structure ͉ phosphoryl transfer ͉ isosteric isoelectronic ͉ transition state analogue P hosphate transfer reactions play a central role in metabolism, regulation, energy housekeeping and signaling (1). As phosphate esters are kinetically extremely stable, efficient catalysis is crucial for the control of these cellular processes. Although model studies have taught us much about the intrinsic chemical mechanisms (2), our understanding of the origins of the enormous enzymatic rate accelerations involved, up to a factor of 10 21 (3), is far from complete (4). A snapshot of an enzyme in a high-energy state would be immensely useful, as it would allow the very interactions that bring about catalysis to be observed (5). However, is this realistic given how elusive high-energy intermediates and transition states (TSs) inevitably are? The direct observation of TSs for simple organic reactions has required ultrafast lasers with femtosecond resolution (6) and no physical or spectroscopic method is available to observe the structure of TSs of enzymatic reactions directly. Thus transition state analogues that bind tightly in an enzyme active site have been of paramount importance in defining the structural and energetic framework for catalysis (7,8).An observation that appears to challenge this paradigm arises from structural studies with ␤-phosphoglucomutase (␤-PGM, EC 5.4.2.6): namely, that a high-energy phosphorane on the reaction pathway has been observed directly by x-ray crystallography, demonstrating how the enzyme interacts with a very high-energy, metastable species (9). The latter also apparently demonstrated that the enzyme catalyzed reaction proceeds through an addition-elimination mechanism, a reaction pathway not observed in solution for phosphate monoester anions. However, the observation of an enzyme "caught in the act" is surprising: the demands of turnover mean that the enzyme would gain no apparent advant...

show abstract

supporting

confidence: 52%

Section: Resultsmentioning

confidence: 74%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

A Trojan horse transition state analogue generated by MgF ₃ ⁻ formation in an enzyme active site

Baxter

Olguín

Goličnik

et al. 2006

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

113

View full text Add to dashboard Cite

show abstract

“…Recently, a crystal structure of an intermediate of an enzyme-catalyzed phosphoryl transfer reaction was reported (20). This intermediate was analyzed as a pentacovalent phosphate structure, but there is considerable controversy concerning this interpretation (21)(22)(23).…”

mentioning

confidence: 99%

A phosphoryl transfer intermediate in the GTPase reaction of Ras in complex with its GTPase-activating protein

Kötting¹,

Blessenohl²,

Suveyzdis³

et al. 2006

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

View full text Add to dashboard Cite

The hydrolysis of nucleoside triphosphates by enzymes is used as a regulation mechanism in key biological processes. Here, the GTP hydrolysis of the protein complex of Ras with its GTPase-activating protein is monitored at atomic resolution in a noncrystalline state by time-resolved FTIR spectroscopy. At 900 ms, after the attack of water at the ␥-phosphate, there appears a H2PO 4 proteins ͉ reaction mechanism ͉ FTIR spectroscopy ͉ signal transduction T he guanine nucleotide-binding protein Ras regulates several signal transduction processes involved in cell growth and differentiation (1). Ras serves as a prototype for the superfamily of GTPases that cycle between the active GTPbound state and the inactive GDP-bound state. The switchingoff of signal transduction is accomplished by GTP hydrolysis, which is a phosphoryl transfer from GTP to water. This process is slow for Ras⅐GTP, allowing further control of this process by GTPase-activating proteins (GAPs), which stimulate the reaction by several orders of magnitude (2). The Ras protein has been extensively studied by various methods, including x-ray crystallography (3-5), NMR (6), computation (7-10), and FTIR spectroscopy (11)(12)(13)(14)(15). For the slow intrinsic reaction, a substrate-assisted mechanism has been inferred from biochemical and computational studies, whereby ␥-phosphate acts as a general base to activate the nucleophilic water (16,17). Accumulating intermediates in GAP-catalyzed reactions have recently been observed for both the Ras⅐RasGAP and the Rap⅐RapGAP reaction, and these intermediates can either decompose to the products GDP and P i or regenerate GTP (11,18,19). Recently, a crystal structure of an intermediate of an enzyme-catalyzed phosphoryl transfer reaction was reported (20). This intermediate was analyzed as a pentacovalent phosphate structure, but there is considerable controversy concerning this interpretation (21-23).Time-resolved FTIR (trFTIR) difference spectroscopy monitors protein reactions at the atomic level in real time (24,25). In the present application, Ras in the presence of saturating amounts of NF1-333, the catalytic GAP domain of neurofibromin, was loaded with caged GTP, which cannot be hydrolyzed by the Ras⅐RasGAP system. Ras⅐GTP was generated by a laser f lash, and the subsequent hydrolysis reaction was monitored by tr-FTIR with a time resolution of milliseconds. The phosphate vibrations were assigned by using 18 Olabeled caged GTP (12). A global multiexponential kinetic analysis of the trFTIR data obtained at 260 K was performed by using a sum of three exponential functions for the GAPcatalyzed GTPase reaction of Ras (11).The first process, with the rate constant k 1 (data not shown), describes the appearance of GTP from the precursor caged GTP (11). At the second rate (rate constant k 2 ), GTP disappears, and an intermediate appears that is assigned to protein-bound P i , initially without implication for its structure. P i is released from the protein in the rate-limiting step, which is described by the third ra...

show abstract

“…The rate and equilibrium for phosphorylation of the active site Asp8 by G16bisP are examined. A model for PGM catalysis is proposed that is based on cycling of the enzyme between the open and closed conformations observed in the reported PGM X-ray crystal structures (6,(12)(13)(14). lactis -phosphoglucomutase ( PGM) was prepared according to a published procedure (16).…”

mentioning

confidence: 99%

Conformational Cycling in β-Phosphoglucomutase Catalysis: Reorientation of the β-d-Glucose 1,6-(Bis)phosphate Intermediate

et al. 2006

Self Cite

View full text Add to dashboard Cite

Activated Lactococcus lactis -phosphoglucomutase ( PGM) catalyzes the conversion of -Dglucose 1-phosphate ( G1P) derived from maltose to -D-glucose 6-phosphate (G6P). Activation requires Mg 2+ binding and phosphorylation of the active site residue Asp8. Initial velocity techniques were used to define the steady-state kinetic constants k cat ) 177 ( 9 s -1 , K m ) 49 ( 4 µM for the substrate G1P and K m ) 6.5 ( 0.7 µM for the activator -D-glucose 1,6-bisphosphate ( G1,6bisP). The observed transient accumulation of [ 14 C] G1,6bisP (12% at ∼0.1 s) in the single turnover reaction carried out with excess PGM (40 µM) and limiting [ 14 C] G1P (5 µM) and G1,6bisP (5 µM) supported the role of G1,6bisP as a reaction intermediate in the conversion of the G1P to G6P. Single turnover reactions of [ 14 C] G1,-6bisP with excess PGM were carried out to demonstrate that phosphoryl transfer rather than ligand binding is rate-limiting and to show that the G1,6bisP binds to the active site in two different orientations (one positioning the C(1)phosphoryl group for reaction with Asp8, and the other orientation positioning the C(6)phosphoryl group for reaction with Asp8) with roughly the same efficiency. Single turnover reactions carried out with PGM, [ 14 C] G1P, and unlabeled G1,6bisP demonstrated complete exchange of label to the G1,6bisP during the catalytic cycle. Thus, the reorientation of the G1,6bisP intermediate that is required to complete the catalytic cycle occurs by diffusion into solvent followed by binding in the opposite orientation. Published X-ray structures of G1P suggest that the reorientation and phosphoryl transfer from G1,6bisP occur by conformational cycling of the enzyme between the active site open and closed forms via cap domain movement. Last, the equilibrium ratio of G1,6bisP to G1P plus G6P was examined to evidence a significant stabilization of PGM aspartyl phosphate.Phosphoglucomutases catalyze the interconversion of D-glucose 1-phosphate (G1P) 1 and D-glucose 6-phosphate (G6P). Operating in the forward G6P-forming direction, this reaction links polysaccharide phosphorolysis to glycolysis. In the reverse direction, the reaction provides G1P for the biosynthesis of exo-polysaccharides (2). There are two classes of phosphoglucomutases, the R-phosphoglucomutases (RPGM, EC 5.4.2.2), ubiquitous among eucaryotes and procaryotes, and the -phosphoglucomutases ( PGM, EC 5.4.2.6), present in certain bacteria and protists. The two classes of mutases are distinguished by their specificity for R-and -D-glucose phosphates and by their protein-fold family. The rabbit muscle RPGM (3) and the closely related Pseudomonas aeruginosa RPGM/RPMM (4) are members of the phosphohexomutase enzyme superfamily (5), whereas PGM (6) belongs to the haloalkanoic acid (HAD) enzyme superfamily (7). The four-domain RPGM and RPGM/RPMM (∼50 kDa) are approximately twice the size of the twodomain PGM (∼25 kDa).In RPGM (and in RPGM/RPMM), phosphoryl transfer is mediated by an active site serine which forms a stable phosphate ester...

show abstract

Chemical Confirmation of a Pentavalent Phosphorane in Complex with β-Phosphoglucomutase

Cited by 57 publications

References 8 publications

A Trojan horse transition state analogue generated by MgF ₃ ⁻ formation in an enzyme active site

A Trojan horse transition state analogue generated by MgF ₃ ⁻ formation in an enzyme active site

A phosphoryl transfer intermediate in the GTPase reaction of Ras in complex with its GTPase-activating protein

Conformational Cycling in β-Phosphoglucomutase Catalysis: Reorientation of the β-d-Glucose 1,6-(Bis)phosphate Intermediate

Contact Info

Product

Resources

About

Chemical Confirmation of a Pentavalent Phosphorane in Complex with β-Phosphoglucomutase

Cited by 57 publications

References 8 publications

A Trojan horse transition state analogue generated by MgF 3 − formation in an enzyme active site

A Trojan horse transition state analogue generated by MgF 3 − formation in an enzyme active site

A phosphoryl transfer intermediate in the GTPase reaction of Ras in complex with its GTPase-activating protein

Conformational Cycling in β-Phosphoglucomutase Catalysis: Reorientation of the β-d-Glucose 1,6-(Bis)phosphate Intermediate

Contact Info

Product

Resources

About

A Trojan horse transition state analogue generated by MgF ₃ ⁻ formation in an enzyme active site

A Trojan horse transition state analogue generated by MgF ₃ ⁻ formation in an enzyme active site