Mechanism and Catalysis of Reactions of Acyl Phosphates. II. Hydrolysis<sup>1</sup>

Sabato, Giovanni Di; Jencks, William P.

doi:10.1021/ja01482a025

Cited by 178 publications

(120 citation statements)

References 6 publications

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“…6, which are published as supporting information on the PNAS web site). This is to be expected from the estimated half-life of a carboxyl phosphate (t 1/2 Ϸ 20 h) for acetylphosphate (20) and is consistent with more recent data on ␤-PGM (10), but differs from the original report (9). However, ␤-PGM also exhibits phosphodismutase activity so the enzyme does not need to be initially phosphorylated nor require the addition of the bisphosphorylated intermediate for mutase activity.…”

Section: Resultssupporting

confidence: 89%

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

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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...

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Section: Resultssupporting

confidence: 89%

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

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“…This behavior differs from that of phosphate monoesters, in which ionization increases reactivity (38 -41) by factors as large as 10 9 (41), and from that of acyl phosphate anhydrides, in which reactivity increases ϳ100-fold in alkaline solutions (42,43).…”

Section: ϫmentioning

confidence: 75%

The Intrinsic Reactivity of ATP and the Catalytic Proficiencies of Kinases Acting on Glucose, N-Acetylgalactosamine, and Homoserine

Stockbridge

Wolfenden

2009

Journal of Biological Chemistry

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To evaluate the rate enhancements produced by representative kinases and their thermodynamic basis, rate constants were determined as a function of changing temperature for 1) the spontaneous methanolysis of ATP and 2) reactions catalyzed by kinases to which different mechanisms of action have been ascribed. A common property shared by enzymes catalyzing biological reactions that involve a single substrate and hydrolytic and hydration reactions in which the effective concentration of water (the second substrate) cannot be elevated much above its concentration in living tissue is their ability to lower the reaction heat of activation. The rates of these enzymatic reactions are less sensitive to temperature than are the rates of the uncatalyzed reactions, so that the rate enhancements and transition state affinities generated by enzymes of this kind increase with decreasing temperature. Entropic effects tend to be minor, with an average change in the entropy of activation that approaches zero (1). That behavior seems understandable in view of the absence of a second substrate or the fact that the second substrate (water) is present in abundance. Thus, approximation effects seem relatively unlikely to play a major role in catalysis by enzymes of those types. Instead, enthalpic effects play a prominent role in catalysis, consistent with the formation in the transition state of new electrostatic and H-bonds between the enzyme and substrate, for which much evidence exists in the structures of enzymes crystallized with transition state analogue inhibitors (1).The effects of enzymes on the enthalpies and entropies of activation of two-substrate reactions remain largely unexplored. The thermodynamics of activation (for an enzyme reaction and for the corresponding uncatalyzed reaction) were examined recently for an unusual two-substrate enzyme (2, 3). The peptidyl transferase center of the ribosome was found to produce a 3 ϫ 10 7 -fold enhancement of the rate of peptidyl ester aminolysis entirely by rendering T⌬S ‡ more favorable, whereas ⌬H ‡ was actually found to become less favorable on the enzyme than in solution (3). Juxtaposition, that is, the gathering of two or more substrates at an enzyme active site in a configuration appropriate for reaction, appears to be partly responsible for that rate enhancement, and desolvation of the substrates may also contribute to the observed rate enhancement (4, 5). Thus, the ribosome might be said to function as a predominantly "physical" catalyst, bringing the substrates near each other in a water-poor environment rather than as a predominantly "chemical" catalyst that participates in the reaction as a general acid or a general base. Consistent with that interpretation is the finding that ribosomal peptidyl transfer is very strongly inhibited by a bisubstrate analogue devised by Yarus and co-workers (6).Because of its unusual composition (consisting entirely of RNA) and the relatively small rate enhancement that it produces, the behavior of the peptidyltransferase center of the r...

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“…In examining the literature on acetyl phosphate hydrolysis, we found that the aspartyl phosphate group might be expected to persist under physiological conditions for several hours. Di Sabato and Jencks (47) reported that the rate of hydrolysis of acetyl phosphate is pH independent between pH 2.5 and 7.5 and that at pH 6.9, 39°C, the t 1/2 is 2.7 h, and at 25°C the t 1/2 is 21 h.…”

Section: Resultsmentioning

confidence: 99%

Caught in the Act: The Structure of Phosphorylated β-Phosphoglucomutase from Lactococcus lactis^,

et al. 2002

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Phosphoglucomutases catalyze the interconversion of D-glucose 1-phosphate and D-glucose 6-phosphate, a reaction central to energy metabolism in all cells and to the synthesis of cell wall polysaccharides in bacterial cells. Two classes of phosphoglucomutases (R-PGM and -PGM) are distinguished on the basis of their specificity for R-and -glucose-1-phosphate. -PGM is a member of the haloacid dehalogenase (HAD) superfamily, which includes the sarcoplasmic Ca 2+ -ATPase, phosphomannomutase, and phosphoserine phosphatase. -PGM is unusual among family members in that the common phosphoenzyme intermediate exists as a stable ground-state complex in this enzyme. Herein we report, for the first time, the three-dimensional structure of a -PGM and the first view of the true phosphoenzyme intermediate in the HAD superfamily. The crystal structure of the Mg(II) complex of phosphorylated -phosphoglucomutase ( -PGM) from Lactococcus lactis has been determined to 2.3 Å resolution by multiwavelength anomalous diffraction (MAD) phasing on selenomethionine, and refined to an R cryst ) 0.24 and R free ) 0.28. The active site of -PGM is located between the core and the cap domain and is freely solvent accessible. The residues within a 6 Å radius of the phosphorylated Asp8 include Asp10, Thr16, Ser114, Lys145, Glu169, and Asp170. The cofactor Mg 2+ is liganded with octahedral coordination geometry by the carboxylate side chains of Asp8, Glu169, Asp170, and the backbone carbonyl oxygen of Asp10 along with one oxygen from the Asp8-phosphoryl group and one water ligand. The phosphate group of the phosphoaspartyl residue, Asp8, interacts with the side chains of Ser114 and Lys145. The absence of a base residue near the aspartyl phosphate group accounts for the persistence of the phosphorylated enzyme under physiological conditions. Substrate docking shows that glucose-6-P can bind to the active site of phosphorylated -PGM in such a way as to position the C(1)OH near the phosphoryl group of the phosphorylated Asp8 and the C(6) phosphoryl group near the carboxylate group of Asp10. This result suggests a novel two-base mechanism for phosphoryl group transfer in a phosphorylated sugar.In this paper, we report the three-dimensional structure of the -phosphoglucomutase from Lactococcus lactis. Both R-and -phosphoglucomutases catalyze the interconversion of D-glucose-1-phosphate (G1P) and glucose-6-phosphate (G6P). This reaction is central to energy metabolism in all cells and is essential to the synthesis of cell wall polysaccharides in bacterial cells (1, 2). The R-phosphoglucomutase (R-PGM) acts on the R-C(1) anomer of G1P, while the -phosphoglucomutase ( -PGM) catalyzes the reaction of the -C(1) anomer. Both mutases employ Mg 2+ and -or R-glucose 1,6-diphosphate (G1,6-diP) as cofactors (1, 2). In addition, both mutases are monomeric proteins. The R-PGM (65 kDa) is, however, approximately twice the size of the -PGM (25 kDa) (3). The X-ray structure of R-PGM from rat reveals a 4-domain R-/ -protein. All four domains contribute residue...

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Mechanism and Catalysis of Reactions of Acyl Phosphates. II. Hydrolysis¹

Cited by 178 publications

References 6 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

The Intrinsic Reactivity of ATP and the Catalytic Proficiencies of Kinases Acting on Glucose, N-Acetylgalactosamine, and Homoserine

Caught in the Act: The Structure of Phosphorylated β-Phosphoglucomutase from Lactococcus lactis^,

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Mechanism and Catalysis of Reactions of Acyl Phosphates. II. Hydrolysis1

Cited by 178 publications

References 6 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

The Intrinsic Reactivity of ATP and the Catalytic Proficiencies of Kinases Acting on Glucose, N-Acetylgalactosamine, and Homoserine

Caught in the Act: The Structure of Phosphorylated β-Phosphoglucomutase from Lactococcus lactis,

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Mechanism and Catalysis of Reactions of Acyl Phosphates. II. Hydrolysis¹

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

Caught in the Act: The Structure of Phosphorylated β-Phosphoglucomutase from Lactococcus lactis^,