Guoqiang Feng

Cleavage and isomerization of UpU promoted by dinuclear metal ion complexes.

The catalysis of phosphoryl transfer by metal ions has been intensively studied in both biological and artificial systems, but the status of the transient pentacoordinate phosphoryl species (as transition state or intermediate) is the subject of considerable debate. We report that dinuclear metal ion complexes that incorporate second sphere hydrogen bond donors not only promote the cleavage of RNA fragments just as efficiently as the activated analogue HPNPP but also provide the first examples of metal ion catalyzed phosphate diester isomerization close to neutral pH. This observation implies that the reaction catalyzed by these complexes involves the formation of a phosphorane intermediate that is sufficiently long-lived to pseudorotate.

A Trojan horse transition state analogue generated by MgF3− formation in an enzyme active site

Identifying how enzymes stabilize high-energy species along the reaction pathway is central to explaining their enormous rate acceleration. β-Phosphoglucomutase catalyses the isomerization of β-glucose-1-phosphate to β-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 19F-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 MgF3− mimics the transferring PO3− 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 β-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.

Mechanistic Study of Protein Phosphatase-1 (PP1), A Catalytically Promiscuous Enzyme

The reaction catalyzed by the protein phosphatase-1 (PP1) has been examined by linear free energy relationships and kinetic isotope effects. With the substrate 4-nitrophenyl phosphate (4NPP), the reaction exhibits a bell-shaped pH-rate profile for kcat/KM indicative of catalysis by both acidic and basic residues, with kinetic pKa values of 6.0 and 7.2. The enzymatic hydrolysis of a series of aryl monoester substrates yields a Brønsted beta(lg) of -0.32, considerably less negative than that of the uncatalyzed hydrolysis of monoester dianions (-1.23). Kinetic isotope effects in the leaving group with the substrate 4NPP are (18)(V/K) bridge = 1.0170 and (15)(V/K) = 1.0010, which, compared against other enzymatic KIEs with and without general acid catalysis, are consistent with a loose transition state with partial neutralization of the leaving group. PP1 also efficiently catalyzes the hydrolysis of 4-nitrophenyl methylphosphonate (4NPMP). The enzymatic hydrolysis of a series of aryl methylphosphonate substrates yields a Brønsted beta(lg) of -0.30, smaller than the alkaline hydrolysis (-0.69) and similar to the beta(lg) measured for monoester substrates, indicative of similar transition states. The KIEs and the beta(lg) data point to a transition state for the alkaline hydrolysis of 4NPMP that is similar to that of diesters with the same leaving group. For the enzymatic reaction of 4NPMP, the KIEs are indicative of a transition state that is somewhat looser than the alkaline hydrolysis reaction and similar to the PP1-catalyzed monoester reaction. The data cumulatively point to enzymatic transition states for aryl phosphate monoester and aryl methylphosphonate hydrolysis reactions that are much more similar to one another than the nonenzymatic hydrolysis reactions of the two substrates.

Comparing a mononuclear Zn(II) complex with hydrogen bond donors with a dinuclear Zn(II) complex for catalysing phosphate ester cleavage

Introducing ligand based hydrogen bond donors to increase the activity of a mononuclear Zn(II) complex for catalysing phosphate ester cleavage can be a more effective strategy than making the dinuclear analogue.

Kinetic analysis of beta-phosphoglucomutase and its inhibition by magnesium fluoride.

The isomerization of beta-glucose-1-phosphate (betaG1P) to beta-glucose-6-phosphate (G6P) catalyzed by beta-phosphoglucomutase (betaPGM) has been examined using steady- and presteady-state kinetic analysis. In the presence of low concentrations of beta-glucose-1,6-bisphosphate (betaG16BP), the reaction proceeds through a Ping Pong Bi Bi mechanism with substrate inhibition (kcat = 65 s(-1), K(betaG1P) = 15 microM, K(betaG16BP) = 0.7 microM, Ki = 122 microM). If alphaG16BP is used as a cofactor, more complex kinetic behavior is observed, but the nonlinear progress curves can be fit to reveal further catalytic parameters (kcat = 74 s(-1), K(betaG1P) = 15 microM, K(betaG16BP) = 0.8 microM, Ki = 122 microM, K(alphaG16BP) = 91 microM for productive binding, K(alphaG16BP) = 21 microM for unproductive binding). These data reveal that variations in the substrate structure affect transition-state affinity (approximately 140,000-fold in terms of rate acceleration) substantially more than ground-state binding (110-fold in terms of binding affinity). When fluoride and magnesium ions are present, time-dependent inhibition of the betaPGM is observed. The concentration dependence of the parameters obtained from fitting these progress curves shows that a betaG1P x MgF3(-) x betaPGM inhibitory complex is formed under the reaction conditions. The overall stability constant for this complex is approximately 2 x 10(-16) M(5) and suggests an affinity of the MgF3(-) moiety to this transition-state analogue (TSA) of < or = 70 nM. The detailed kinetic analysis shows how a special type of TSA that does not exist in solution is assembled in the active site of an enzyme. Further experiments show that under the conditions of previous structural studies, phosphorylated glucose only persists when bound to the enzyme as the TSA. The preference for TSA formation when fluoride is present, and the hydrolysis of substrates when it is not, rules out the formation of a stable pentavalent phosphorane intermediate in the active site of betaPGM.

Mechanism and Transition State Structure of Aryl Methylphosphonate Esters Doubly Coordinated to a Dinuclear Cobalt(III) Center

Reactivities of five phosphonate esters each coordinated to a dinuclear Co(III) complex were investigated ([Co(2)(tacn)(2)(OH)(2){O(2)P(Me)OAr}](3+); tacn = 1,4,7-triazacyclononane; substituent = m-F, p-NO(2) (1a); p-NO(2) (1b); m-NO(2) (1c); p-Cl (1d); unsubstituted (1e)). Hydrolysis of the phosphonate esters in 1a to 1e is specific base catalyzed and takes place by intramolecular oxide attack on the bridging phosphonate. These data define a Brønsted beta(lg) of -1.12, considerably more negative than that of the hydrolysis of the uncomplexed phosphonates (-0.69). For 1b, the kinetic isotope effects in the leaving group are (18)k(lg) = 1.0228 and (15)k = 1.0014, at the nonbridging phosphoryl oxygens (18)k(nonbridge) = 0.9954, and at the nucleophilic oxygen(18)k(nuc) = 1.0105. The KIEs and the beta(lg) data point to a transition state for the alkaline hydrolysis of 1b that is similar to that of a phosphate monoester complex with the same leaving group, rather than the isoelectronic diester complex. The data from these model systems parallel the observation that in protein phosphatase-1, which has an active site that resembles the structures of these complexes, the catalyzed hydrolysis of aryl methylphosphonates and aryl phosphates are much more similar to one another than the uncomplexed hydrolysis reactions of the two substrates.

Phosphate ester analogues as probes for understanding enzyme catalysed phosphoryl transfer

Significant research effort has been invested into elucidating the mechanism and reactivity of phosphate mono- and diesters, and how their reactions are efficiently catalysed by enzymes. Although both reactions involve the transfer of a phosphoryl group with similar geometries, it is uncommon to find enzymes capable of catalyzing both reactions and the factors governing the selectivity of this enzyme remain poorly understood. Herein, we examine the reactivity of a series of aryl fluorophosphates to study the effect of changing the nature of the transition state while retaining the size and hydrogen bond donor/acceptor properties of the phosphoryl group in the corresponding monoester. We have performed an extensive kinetic and theoretical analysis of the hydrolysis of the reference reaction in solution, demonstrating that fluorophosphates mimic the behaviour of the corresponding methyl aryl phosphate diesters, proceeding through concerted transition states with structures that are sensitive to the acidity of the leaving group. We have also performed an initial study of the catalysis of this reaction by the R166A mutant of alkaline phosphatase, and find that the enzyme has a greater proficiency with the diester substrate than fluorophosphate substrates, although both are greatly reduced relative to the corresponding monoester.

Photo-induced molecular-recognition-mediated adhesion of giant vesicles.

Few methods currently exist for controlling vesicle-vesicle adhesion. We now report a new system, based upon a multivalent guest and an amphiphilic receptor with a photo-isomerisable anchor that can be incorporated into lipid vesicles of different sizes. Large unilamellar vesicles containing our receptor were found to aggregate upon addition of the multivalent guest, independently of photoswitching between the two conformations of the anchor. However, for giant vesicles immobilised on a platinum wire, guest-mediated adhesion only occurred upon photo-isomerisation of the anchor. This behaviour was attributed to the dynamics introduced into the system through the conformational changes caused by irradiation.