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Alvan C. Hengge - One of the best experts on this subject based on the ideXlab platform.

  • transition state interactions in a promiscuous enzyme sulfate and Phosphate Monoester hydrolysis by pseudomonas aeruginosa arylsulfatase
    Biochemistry, 2019
    Co-Authors: Bert Van Loo, Alvan C. Hengge, Usa Boonyuen, Mark F. Mohamed, Marko Golicnik, Ryan D Berry, Florian Hollfelder
    Abstract:

    Pseudomonas aeruginosa arylsulfatase (PAS) hydrolyzes sulfate and, promiscuously, Phosphate Monoesters. Enzyme-catalyzed sulfate transfer is crucial to a wide variety of biological processes, but detailed studies of the mechanistic contributions to its catalysis are lacking. We present linear free energy relationships (LFERs) and kinetic isotope effects (KIEs) of PAS and analyses of active site mutants that suggest a key role for leaving group (LG) stabilization. In LFERs PASWT has a much less negative Bronsted coefficient (βleaving groupobs-Enz = −0.33) than the uncatalyzed reaction (βleaving groupobs = −1.81). This situation is diminished when cationic active site groups are exchanged for alanine. The considerable degree of bond breaking during the transition state (TS) is evidenced by an 18Obridge KIE of 1.0088. LFER and KIE data for several active site mutants point to leaving group stabilization by active site K375, in cooperation with H211. 15N KIEs and the increased sensitivity to leaving group abi...

  • Transition-State Interactions in a Promiscuous Enzyme: Sulfate and Phosphate Monoester Hydrolysis by Pseudomonas aeruginosa Arylsulfatase
    2019
    Co-Authors: Bert Van Loo, Alvan C. Hengge, Ryan Berry, Usa Boonyuen, Mark F. Mohamed, Marko Golicnik, Florian Hollfelder
    Abstract:

    Pseudomonas aeruginosa arylsulfatase (PAS) hydrolyzes sulfate and, promiscuously, Phosphate Monoesters. Enzyme-catalyzed sulfate transfer is crucial to a wide variety of biological processes, but detailed studies of the mechanistic contributions to its catalysis are lacking. We present linear free energy relationships (LFERs) and kinetic isotope effects (KIEs) of PAS and analyses of active site mutants that suggest a key role for leaving group (LG) stabilization. In LFERs PASWT has a much less negative Brønsted coefficient (βleaving groupobs‑Enz = −0.33) than the uncatalyzed reaction (βleaving groupobs = −1.81). This situation is diminished when cationic active site groups are exchanged for alanine. The considerable degree of bond breaking during the transition state (TS) is evidenced by an 18Obridge KIE of 1.0088. LFER and KIE data for several active site mutants point to leaving group stabilization by active site K375, in cooperation with H211. 15N KIEs and the increased sensitivity to leaving group ability of the sulfatase activity in neat D2O (Δβleaving groupH‑D = +0.06) suggest that the mechanism for S–Obridge bond fission shifts, with decreasing leaving group ability, from charge compensation via Lewis acid interactions toward direct proton donation. 18Ononbridge KIEs indicate that the TS for PAS-catalyzed sulfate Monoester hydrolysis has a significantly more associative character compared to the uncatalyzed reaction, while PAS-catalyzed Phosphate Monoester hydrolysis does not show this shift. This difference in enzyme-catalyzed TSs appears to be the major factor favoring specificity toward sulfate over Phosphate esters by this promiscuous hydrolase, since other features are either too similar (uncatalyzed TS) or inherently favor Phosphate (charge)

  • Transition State Interactions in a Promiscuous Enzyme: Sulfate and Phosphate Monoester Hydrolysis by Pseudomonas aeruginosa Arylsulfatase
    2018
    Co-Authors: Bert Van Loo, Alvan C. Hengge, Ryan Berry, Usa Boonyuen, Mark F. Mohamed, Marko Golicnik, Florian Hollfelder
    Abstract:

    Pseudomonas aeruginosa arylsulfatase (PAS) hydrolyses sulfate and, promiscuously, Phosphate Monoesters. Enzyme-catalyzed sulfate transfer is crucial to a wide variety of biological processes, but detailed studies of the mechanistic contributions to its catalysis are lacking. We present an investigation based on linear free energy relationships (LFERs) and kinetic isotope effects (KIEs) of PAS and active site mutants that suggest a key role for leaving group (LG) stabilization. In LFERs wild type PAS has a much less negative Bronsted coefficient (βleaving group/obs-Enz = -0.33) than the uncatalyzed reaction (βleaving group/obs = -1.81). This situation is diminished when cationic active site groups are exchanged for alanine. The considerable degree of bond breaking during the TS is evidenced by an 18Obridge KIE of 1.0088. LFER and KIE data for several active site mutants point to leaving group stabilization by active-site lysine K375, in cooperation with histidine H211. 15N KIEs combined with an increased sensitivity to leaving group ability of the sulfatase activity in neat D2O (Δβleaving group H-D = +0.06) suggest that the mechanism for S-Obridge bond fission shifts, with decreasing leaving group ability, from charge compensation via Lewis acid interactions towards direct proton donation. 18Ononbridge KIEs indicate that the TS for PAScatalyzed sulfate Monoester hydrolysis has a significantly more associative character compared to the uncatalyzed reaction, while PAS-catalyzed Phosphate Monoester hydrolysis does not show this shift. This difference in enzyme-catalyzed TSs appears to be the major factor favoring specificity toward sulfate over Phosphate in this promiscuous hydrolase, since other features are either too similar (uncatalyzed TS) or inherently favor Phosphate (charge).

  • mechanism and transition state structure of aryl methylphosphonate esters doubly coordinated to a dinuclear cobalt iii center
    Journal of the American Chemical Society, 2009
    Co-Authors: Guoqiang Feng, Eric A Tanifum, Alvan C. Hengge, Harry Adams, Nicholas H Williams
    Abstract:

    Reactivities of five phosphonate esters each coordinated to a dinuclear Co(III) complex were investigated ([Co2(tacn)2(OH)2{O2P(Me)OAr}]3+; tacn = 1,4,7-triazacyclononane; substituent = m-F, p-NO2 (1a); p-NO2 (1b); m-NO2 (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 Bronsted βlg 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 18klg = 1.0228 and 15k = 1.0014, at the nonbridging phosphoryl oxygens 18knonbridge = 0.9954, and at the nucleophilic oxygen18knuc = 1.0105. The KIEs and the βlg 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 ...

  • Kinetic Isotope Effects for Alkaline Phosphatase Reactions: Implications for the Role of Active-Site Metal Ions in Catalysis
    Journal of the American Chemical Society, 2007
    Co-Authors: Jesse G. Zalatan, Daniel Herschlag, Irina E. Catrina, Rebecca Mitchell, Piotr K. Grzyska, Patrick J. O'brien, Alvan C. Hengge
    Abstract:

    Enzyme-catalyzed phosphoryl transfer reactions have frequently been suggested to proceed through transition states that are altered from their solution counterparts, with the alterations presumably arising from interactions with active-site functional groups. In particular, the Phosphate Monoester hydrolysis reaction catalyzed by Escherichia coli alkaline phosphatase (AP) has been the subject of intensive scrutiny. Recent linear free energy relationship (LFER) studies suggest that AP catalyzes Phosphate Monoester hydrolysis through a loose transition state, similar to that in solution. To gain further insight into the nature of the transition state and active-site interactions, we have determined kinetic isotope effects (KIEs) for AP-catalyzed hydrolysis reactions with several Phosphate Monoester substrates. The LFER and KIE data together provide a consistent picture for the nature of the transition state for AP-catalyzed Phosphate Monoester hydrolysis and support previous models suggesting that the enzym...

Alain Vigroux - One of the best experts on this subject based on the ideXlab platform.

  • Theoretical evaluation of the substrate-assisted catalysis mechanism for the hydrolysis of Phosphate Monoester dianions.
    Chemistry - A European Journal, 2007
    Co-Authors: Nathalie Iché‐tarrat, Manuel F. Ruiz-lópez, Jean-claude Barthelat, Alain Vigroux
    Abstract:

    Quantum chemistry methods coupled with a continuum solvation model have been applied to evaluate the substrate-assisted catalysis (SAC) mechanism recently proposed for the hydrolysis of Phosphate Monoester dianions. The SAC mechanism, in which a proton from the nucleophile is transferred to a nonbridging phosphoryl oxygen atom of the substrate prior to attack, has been proposed in opposition to the widely accepted mechanism of direct nucleophilic reaction. We have assessed the SAC proposal for the hydrolysis of three representative Phosphate Monoester dianions (2,4-dinitrophenyl Phosphate, phenyl Phosphate, and methyl Phosphate) by considering the reactivity of the hydroxide ion toward the phosphorus center of the corresponding singly protonated Monoesters. The reliability of the calculations was verified by comparing the calculated and the observed values of the activation free energies for the analogous S(N)2(P) reactions of F- with the monoanion of the Monoester 2,4-dinitrophenyl Phosphate and its diester analogue, methyl 2,4-dinitrophenyl Phosphate. It was found that the orientation of the Phosphate hydrogen atom has important implications with regard to the nature of the transition state. Hard nucleophiles such as OH- and F- can attack the phosphorus atom of a singly protonated Phosphate Monoester only if the Phosphate hydrogen atom is oriented toward the leaving-group oxygen atom. As a result of this proton orientation, the SAC mechanism in solution is characterized by a small Bronsted coefficient value (beta(lg)=-0.25). This mechanism is unlikely to apply to aryl Phosphates, but becomes a likely possibility for alkyl Phosphate esters. If oxyanionic nucleophiles of pK(a)

  • theoretical evaluation of the substrate assisted catalysis mechanism for the hydrolysis of Phosphate Monoester dianions
    Chemistry: A European Journal, 2007
    Co-Authors: Nathalie Ichetarrat, Jean-claude Barthelat, Manuel F Ruizlopez, Alain Vigroux
    Abstract:

    Quantum chemistry methods coupled with a continuum solvation model have been applied to evaluate the substrate-assisted catalysis (SAC) mechanism recently proposed for the hydrolysis of Phosphate Monoester dianions. The SAC mechanism, in which a proton from the nucleophile is transferred to a nonbridging phosphoryl oxygen atom of the substrate prior to attack, has been proposed in opposition to the widely accepted mechanism of direct nucleophilic reaction. We have assessed the SAC proposal for the hydrolysis of three representative Phosphate Monoester dianions (2,4-dinitrophenyl Phosphate, phenyl Phosphate, and methyl Phosphate) by considering the reactivity of the hydroxide ion toward the phosphorus center of the corresponding singly protonated Monoesters. The reliability of the calculations was verified by comparing the calculated and the observed values of the activation free energies for the analogous S(N)2(P) reactions of F- with the monoanion of the Monoester 2,4-dinitrophenyl Phosphate and its diester analogue, methyl 2,4-dinitrophenyl Phosphate. It was found that the orientation of the Phosphate hydrogen atom has important implications with regard to the nature of the transition state. Hard nucleophiles such as OH- and F- can attack the phosphorus atom of a singly protonated Phosphate Monoester only if the Phosphate hydrogen atom is oriented toward the leaving-group oxygen atom. As a result of this proton orientation, the SAC mechanism in solution is characterized by a small Bronsted coefficient value (beta(lg)=-0.25). This mechanism is unlikely to apply to aryl Phosphates, but becomes a likely possibility for alkyl Phosphate esters. If oxyanionic nucleophiles of pK(a)<11 are involved, as in alkaline phosphatase, then the S(N)2(P) reaction may proceed with the Phosphate hydrogen atom oriented toward the nucleophile. In this situation, a large negative value of beta(lg) (-0.95) is predicted for the substrate-assisted catalysis mechanism.

Daniel Herschlag - One of the best experts on this subject based on the ideXlab platform.

  • Probing the Origins of Catalytic Discrimination between Phosphate and Sulfate Monoester Hydrolysis: Comparative Analysis of Alkaline
    2016
    Co-Authors: Protein Tyrosine Phosphatases, Jesse G. Zalatan, Logan D. Andrews, Daniel Herschlag
    Abstract:

    ABSTRACT: Catalytic promiscuity, the ability of enzymes to catalyze multiple reactions, provides an opportunity to gain a deeper understanding of the origins of catalysis and substrate specificity. Alkaline phosphatase (AP) catalyzes both phos-phate and sulfate Monoester hydrolysis reactions with a ∼1010-fold preference for Phosphate Monoester hydrolysis, despite the similarity between these reactions. The preponderance of formal positive charge in the AP active site, particularly from three divalent metal ions, was proposed to be responsible for this preference by providing stronger electrostatic interactions with the more negatively charged phosphoryl group versus the sulfuryl group. To test whether positively charged metal ions are required to achieve a high preference for the Phosphate Monoester hydrolysis reaction, the catalytic preference of three protein tyrosine phosphatases (PTPs), which do not contain metal ions, were measured. Their preferences ranged from 5 × 106 to 7 × 107, lower than that for AP but still substantial, indicating that metal ions and a high preponderance of formal positive charge within the active site are not required to achieve a strong catalytic preference for Phosphate Monoester over sulfate Monoester hydrolysis. The observed ionic strength dependence

  • Probing the Origins of Catalytic Discrimination between Phosphate and Sulfate Monoester Hydrolysis: Comparative Analysis of Alkaline Phosphatase and Protein Tyrosine Phosphatases
    2015
    Co-Authors: Logan D. Andrews, Jesse G. Zalatan, Daniel Herschlag
    Abstract:

    Catalytic promiscuity, the ability of enzymes to catalyze multiple reactions, provides an opportunity to gain a deeper understanding of the origins of catalysis and substrate specificity. Alkaline phosphatase (AP) catalyzes both Phosphate and sulfate Monoester hydrolysis reactions with a ∼1010-fold preference for Phosphate Monoester hydrolysis, despite the similarity between these reactions. The preponderance of formal positive charge in the AP active site, particularly from three divalent metal ions, was proposed to be responsible for this preference by providing stronger electrostatic interactions with the more negatively charged phosphoryl group versus the sulfuryl group. To test whether positively charged metal ions are required to achieve a high preference for the Phosphate Monoester hydrolysis reaction, the catalytic preference of three protein tyrosine phosphatases (PTPs), which do not contain metal ions, were measured. Their preferences ranged from 5 × 106 to 7 × 107, lower than that for AP but still substantial, indicating that metal ions and a high preponderance of formal positive charge within the active site are not required to achieve a strong catalytic preference for Phosphate Monoester over sulfate Monoester hydrolysis. The observed ionic strength dependences of kcat/KM values for Phosphate and sulfate Monoester hydrolysis are steeper for the more highly charged Phosphate ester with both AP and the PTP Stp1, following the dependence expected based on the charge difference of these two substrates. However, the dependences for AP were not greater than those of Stp1 and were rather shallow for both enzymes. These results suggest that overall electrostatics from formal positive charge within the active site is not the major driving force in distinguishing between these reactions and that substantial discrimination can be attained without metal ions. Thus, local properties of the active site, presumably including multiple positioned dipolar hydrogen bond donors within the active site, dominate in defining this reaction specificity

  • probing the origins of catalytic discrimination between Phosphate and sulfate Monoester hydrolysis comparative analysis of alkaline phosphatase and protein tyrosine phosphatases
    Biochemistry, 2014
    Co-Authors: Logan D. Andrews, Jesse G. Zalatan, Daniel Herschlag
    Abstract:

    Catalytic promiscuity, the ability of enzymes to catalyze multiple reactions, provides an opportunity to gain a deeper understanding of the origins of catalysis and substrate specificity. Alkaline phosphatase (AP) catalyzes both Phosphate and sulfate Monoester hydrolysis reactions with a ∼1010-fold preference for Phosphate Monoester hydrolysis, despite the similarity between these reactions. The preponderance of formal positive charge in the AP active site, particularly from three divalent metal ions, was proposed to be responsible for this preference by providing stronger electrostatic interactions with the more negatively charged phosphoryl group versus the sulfuryl group. To test whether positively charged metal ions are required to achieve a high preference for the Phosphate Monoester hydrolysis reaction, the catalytic preference of three protein tyrosine phosphatases (PTPs), which do not contain metal ions, were measured. Their preferences ranged from 5 × 106 to 7 × 107, lower than that for AP but st...

  • Kinetic Isotope Effects for Alkaline Phosphatase Reactions: Implications for the Role of Active-Site Metal Ions in Catalysis
    Journal of the American Chemical Society, 2007
    Co-Authors: Jesse G. Zalatan, Daniel Herschlag, Irina E. Catrina, Rebecca Mitchell, Piotr K. Grzyska, Patrick J. O'brien, Alvan C. Hengge
    Abstract:

    Enzyme-catalyzed phosphoryl transfer reactions have frequently been suggested to proceed through transition states that are altered from their solution counterparts, with the alterations presumably arising from interactions with active-site functional groups. In particular, the Phosphate Monoester hydrolysis reaction catalyzed by Escherichia coli alkaline phosphatase (AP) has been the subject of intensive scrutiny. Recent linear free energy relationship (LFER) studies suggest that AP catalyzes Phosphate Monoester hydrolysis through a loose transition state, similar to that in solution. To gain further insight into the nature of the transition state and active-site interactions, we have determined kinetic isotope effects (KIEs) for AP-catalyzed hydrolysis reactions with several Phosphate Monoester substrates. The LFER and KIE data together provide a consistent picture for the nature of the transition state for AP-catalyzed Phosphate Monoester hydrolysis and support previous models suggesting that the enzym...

  • Functional Interrelationships in the Alkaline Phosphatase Superfamily: Phosphodiesterase Activity of Escherichia coli Alkaline Phosphatase†
    Biochemistry, 2001
    Co-Authors: Patrick J. O'brien, Daniel Herschlag
    Abstract:

    Escherichia coli alkaline phosphatase (AP) is a proficient phosphoMonoesterase with two Zn(2+) ions in its active site. Sequence homology suggests a distant evolutionary relationship between AP and alkaline phosphodiesterase/nucleotide pyrophosphatase, with conservation of the catalytic metal ions. Furthermore, many other phosphodiesterases, although not evolutionarily related, have a similar active site configuration of divalent metal ions in their active sites. These observations led us to test whether AP could also catalyze the hydrolysis of Phosphate diesters. The results described herein demonstrate that AP does have phosphodiesterase activity: the phosphatase and phosphodiesterase activities copurify over several steps; inorganic Phosphate, a strong competitive inhibitor of AP, inhibits the phosphodiesterase and phosphatase activities with the same inhibition constant; a point mutation that weakens Phosphate binding to AP correspondingly weakens Phosphate inhibition of the phosphodiesterase activity; and mutation of active site residues substantially reduces both the mono- and diesterase activities. AP accelerates the rate of Phosphate diester hydrolysis by 10(11)-fold relative to the rate of the uncatalyzed reaction [(k(cat)/K(m))/k(w)]. Although this rate enhancement is substantial, it is at least 10(6)-fold less than the rate enhancement for AP-catalyzed Phosphate Monoester hydrolysis. Mutational analysis suggests that common active site features contribute to hydrolysis of both Phosphate Monoesters and Phosphate diesters. However, mutation of the active site arginine to serine, R166S, decreases the Monoesterase activity but not the diesterase activity, suggesting that the interaction of this arginine with the nonbridging oxygen(s) of the Phosphate Monoester substrate provides a substantial amount of the preferential hydrolysis of Phosphate Monoesters. The observation of phosphodiesterase activity extends the previous observation that AP has a low level of sulfatase activity, further establishing the functional interrelationships among the sulfatases, phosphatases, and phosphodiesterases within the evolutionarily related AP superfamily. The catalytic promiscuity of AP could have facilitated divergent evolution via gene duplication by providing a selective advantage upon which natural selection could have acted.

Nathalie Ichetarrat - One of the best experts on this subject based on the ideXlab platform.

  • theoretical evaluation of the substrate assisted catalysis mechanism for the hydrolysis of Phosphate Monoester dianions
    Chemistry: A European Journal, 2007
    Co-Authors: Nathalie Ichetarrat, Jean-claude Barthelat, Manuel F Ruizlopez, Alain Vigroux
    Abstract:

    Quantum chemistry methods coupled with a continuum solvation model have been applied to evaluate the substrate-assisted catalysis (SAC) mechanism recently proposed for the hydrolysis of Phosphate Monoester dianions. The SAC mechanism, in which a proton from the nucleophile is transferred to a nonbridging phosphoryl oxygen atom of the substrate prior to attack, has been proposed in opposition to the widely accepted mechanism of direct nucleophilic reaction. We have assessed the SAC proposal for the hydrolysis of three representative Phosphate Monoester dianions (2,4-dinitrophenyl Phosphate, phenyl Phosphate, and methyl Phosphate) by considering the reactivity of the hydroxide ion toward the phosphorus center of the corresponding singly protonated Monoesters. The reliability of the calculations was verified by comparing the calculated and the observed values of the activation free energies for the analogous S(N)2(P) reactions of F- with the monoanion of the Monoester 2,4-dinitrophenyl Phosphate and its diester analogue, methyl 2,4-dinitrophenyl Phosphate. It was found that the orientation of the Phosphate hydrogen atom has important implications with regard to the nature of the transition state. Hard nucleophiles such as OH- and F- can attack the phosphorus atom of a singly protonated Phosphate Monoester only if the Phosphate hydrogen atom is oriented toward the leaving-group oxygen atom. As a result of this proton orientation, the SAC mechanism in solution is characterized by a small Bronsted coefficient value (beta(lg)=-0.25). This mechanism is unlikely to apply to aryl Phosphates, but becomes a likely possibility for alkyl Phosphate esters. If oxyanionic nucleophiles of pK(a)<11 are involved, as in alkaline phosphatase, then the S(N)2(P) reaction may proceed with the Phosphate hydrogen atom oriented toward the nucleophile. In this situation, a large negative value of beta(lg) (-0.95) is predicted for the substrate-assisted catalysis mechanism.

Nicholas H Williams - One of the best experts on this subject based on the ideXlab platform.

  • The Competing Mechanisms of Phosphate Monoester Dianion Hydrolysis
    Journal of the American Chemical Society, 2016
    Co-Authors: Fernanda Duarte, Nicholas H Williams, Alexandre Barrozo, Johan Åqvist, Shina Caroline Lynn Kamerlin
    Abstract:

    Despite the numerous experimental and theoretical studies on Phosphate Monoester hydrolysis, significant questions remain concerning the mechanistic details of these biologically critical reactions. In the present work we construct a linear free energy relationship for Phosphate Monoester hydrolysis to explore the effect of modulating leaving group pKa on the competition between solvent- and substrate-assisted pathways for the hydrolysis of these compounds. Through detailed comparative electronic-structure studies of methyl Phosphate and a series of substituted aryl Phosphate Monoesters, we demonstrate that the preferred mechanism is dependent on the nature of the leaving group. For good leaving groups, a strong preference is observed for a more dissociative solvent-assisted pathway. However, the energy difference between the two pathways gradually reduces as the leaving group pKa increases and creates mechanistic ambiguity for reactions involving relatively poor alkoxy leaving groups. Our calculations sh...

  • The Competing Mechanisms of Phosphate Monoester Dianion Hydrolysis
    2016
    Co-Authors: Fernanda Duarte, Nicholas H Williams, Alexandre Barrozo, Johan Åqvist, Shina Caroline Lynn Kamerlin
    Abstract:

    Despite the numerous experimental and theoretical studies on Phosphate Monoester hydrolysis, significant questions remain concerning the mechanistic details of these biologically critical reactions. In the present work we construct a linear free energy relationship for Phosphate Monoester hydrolysis to explore the effect of modulating leaving group pKa on the competition between solvent- and substrate-assisted pathways for the hydrolysis of these compounds. Through detailed comparative electronic-structure studies of methyl Phosphate and a series of substituted aryl Phosphate Monoesters, we demonstrate that the preferred mechanism is dependent on the nature of the leaving group. For good leaving groups, a strong preference is observed for a more dissociative solvent-assisted pathway. However, the energy difference between the two pathways gradually reduces as the leaving group pKa increases and creates mechanistic ambiguity for reactions involving relatively poor alkoxy leaving groups. Our calculations show that the transition-state structures vary smoothly across the range of pKas studied and that the pathways remain discrete mechanistic alternatives. Therefore, while not impossible, a biological catalyst would have to surmount a significantly higher activation barrier to facilitate a substrate-assisted pathway than for the solvent-assisted pathway when Phosphate is bonded to good leaving groups. For poor leaving groups, this intrinsic preference disappears

  • Mechanistic Shifts Along the Linear Free Energy Relationship for Aryl Phosphate Monoester Hydrolysis
    2016
    Co-Authors: Fernanda Duarte, Nicholas H Williams, Alexandre Barrozo, Johan Åqvist, Shina Caroline Lynn Kamerlin
    Abstract:

    Phosphoryl transfers are essential chemical reactions in key life processes, including energy production, signal transduction and protein synthesis. They are known for having extremely low reaction rates in aqueous solution, reaching the scale of millions of years. In order to make life possible, enzymes that catalyse phosphoryl transfer, phosphoryl transferases, have evolved to be tremendously proficient catalysts, increasing reaction rates to the millisecond timescale.Due to the nature of the electronic structure of phosphorus atoms, understanding how hydrolysis of Phosphate esters occurs is a complex task. Experimental studies on the hydrolysis of Phosphate Monoesters with acidic leaving groups suggest a concerted mechanism with a loose, metaPhosphate-like transition state. Theoretical studies have suggested two possible concerted pathways, either with loose or tight transition state geometries, plus the possibility of a stepwise mechanism with the formation of a phosphorane intermediate. Different pathways were shown to be energetically preferable depending on the acidity of the leaving group. Here we performed computational studies to revisit how this mechanistic shift occurs along a series of aryl Phosphate Monoesters, suggesting possible factors leading to such change.The fact that distinct pathways can occur in solution could mean that the same is possible for an enzyme active site. We performed simulations on the catalytic activity of β-phosphoglucomutase, suggesting that it is possible for two mechanisms to occur at the same time for the phosphoryl transfer.Curiously, several phosphoryl transferases were shown to be able to catalyse not only Phosphate ester hydrolysis, but also the cleavage of other compounds. We modeled the catalytic mechanism of two highly promiscuous members of the alkaline phosphatase superfamily. Our model reproduces key experimental observables and shows that these enzymes are electrostatically flexible, employing the same set of residues to enhance the rates of different reactions, with different electrostatic contributions per residue.

  • Resolving Apparent Conflicts between Theoretical and Experimental Models of Phosphate Monoester Hydrolysis
    2015
    Co-Authors: Fernanda Duarte, Nicholas H Williams, Johan Åqvist, Shina C. L. Kamerlin
    Abstract:

    Understanding phosphoryl and sulfuryl transfer is central to many biochemical processes. However, despite decades of experimental and computational studies, a consensus concerning the precise mechanistic details of these reactions has yet to be reached. In this work we perform a detailed comparative theoretical study of the hydrolysis of p-nitrophenyl Phosphate, methyl Phosphate and p-nitrophenyl sulfate, all of which have served as key model systems for understanding phosphoryl and sulfuryl transfer reactions, respectively. We demonstrate the existence of energetically similar but mechanistically distinct possibilities for Phosphate Monoester hydrolysis. The calculated kinetic isotope effects for p-nitrophenyl Phosphate provide a means to discriminate between substrate- and solvent-assisted pathways of Phosphate Monoester hydrolysis, and show that the solvent-assisted pathway dominates in solution. This preferred mechanism for p-nitrophenyl Phosphate hydrolysis is difficult to find computationally due to the limitations of compressing multiple bonding changes onto a 2-dimensional energy surface. This problem is compounded by the need to include implicit solvation to at least microsolvate the system and stabilize the highly charged species. In contrast, methyl Phosphate hydrolysis shows a preference for a substrate-assisted mechanism. For p-nitrophenyl sulfate hydrolysis there is only one viable reaction pathway, which is similar to the solvent-assisted pathway for Phosphate hydrolysis, and the substrate-assisted pathway is not accessible. Overall, our results provide a unifying mechanistic framework that is consistent with the experimentally measured kinetic isotope effects and reconciles the discrepancies between theoretical and experimental models for these biochemically ubiquitous classes of reaction

  • Resolving apparent conflicts between theoretical and experimental models of Phosphate Monoester hydrolysis.
    Journal of the American Chemical Society, 2014
    Co-Authors: Fernanda Duarte, Nicholas H Williams, Johan Åqvist, Shina Caroline Lynn Kamerlin
    Abstract:

    Understanding phosphoryl and sulfuryl transfer is central to many biochemical processes. However, despite decades of experimental and computational studies, a consensus concerning the precise mechanistic details of these reactions has yet to be reached. In this work we perform a detailed comparative theoretical study of the hydrolysis of p-nitrophenyl Phosphate, methyl Phosphate and p-nitrophenyl sulfate, all of which have served as key model systems for understanding phosphoryl and sulfuryl transfer reactions, respectively. We demonstrate the existence of energetically similar but mechanistically distinct possibilities for Phosphate Monoester hydrolysis. The calculated kinetic isotope effects for p-nitrophenyl Phosphate provide a means to discriminate between substrate- and solvent-assisted pathways of Phosphate Monoester hydrolysis, and show that the solvent-assisted pathway dominates in solution. This preferred mechanism for p-nitrophenyl Phosphate hydrolysis is difficult to find computationally due to the limitations of compressing multiple bonding changes onto a 2-dimensional energy surface. This problem is compounded by the need to include implicit solvation to at least microsolvate the system and stabilize the highly charged species. In contrast, methyl Phosphate hydrolysis shows a preference for a substrate-assisted mechanism. For p-nitrophenyl sulfate hydrolysis there is only one viable reaction pathway, which is similar to the solvent-assisted pathway for Phosphate hydrolysis, and the substrate-assisted pathway is not accessible. Overall, our results provide a unifying mechanistic framework that is consistent with the experimentally measured kinetic isotope effects and reconciles the discrepancies between theoretical and experimental models for these biochemically ubiquitous classes of reaction.