Kinetic Isotope Effects for Alkaline Phosphatase Reactions:  Implications for the Role of Active-Site Metal Ions in Catalysis

Zalatan, Jesse G.; Catrina, Irina E.; Mitchell, Rebecca; Grzyska, Piotr K.; O’Brien, Patrick; Herschlag, Daniel; Hengge, Alvan C.

doi:10.1021/ja072196+

Cited by 68 publications

(147 citation statements)

References 81 publications

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“…These results are consistent with a dissociative transition state both on and off the enzyme. However, a more inverse nonbridging oxygen KIE is observed for AP and is consistent with active site metal coordination in the transition state (29). In solution, acid-catalyzed hydrolysis of nucleoside glycosidic bonds occurs via dissociative transition states with considerable oxycarbonium ion character in the ribose ring (31,32).…”

Section: Simulations and Qm Calculations To Model The Mechanism Andsupporting

confidence: 63%

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Experimental and computational analysis of the transition state for ribonuclease A-catalyzed RNA 2′- O -transphosphorylation

Gu¹,

Zhang²,

Wong

et al. 2013

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

125

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Enzymes function by stabilizing reaction transition states; therefore, comparison of the transition states of enzymatic and nonenzymatic model reactions can provide insight into biological catalysis. Catalysis of RNA 2′-O-transphosphorylation by ribonuclease A is proposed to involve electrostatic stabilization and acid/ base catalysis, although the structure of the rate-limiting transition state is uncertain. Here, we describe coordinated kinetic isotope effect (KIE) analyses, molecular dynamics simulations, and quantum mechanical calculations to model the transition state and mechanism of RNase A. Comparison of the 18 O KIEs on the 2′O nucleophile, 5′O leaving group, and nonbridging phosphoryl oxygens for RNase A to values observed for hydronium-or hydroxide-catalyzed reactions indicate a late anionic transition state. Molecular dynamics simulations using an anionic phosphorane transition state mimic suggest that H-bonding by protonated His12 and Lys41 stabilizes the transition state by neutralizing the negative charge on the nonbridging phosphoryl oxygens. Quantum mechanical calculations consistent with the experimental KIEs indicate that expulsion of the 5′O remains an integral feature of the rate-limiting step both on and off the enzyme. Electrostatic interactions with positively charged amino acid site chains (His12/ Lys41), together with proton transfer from His119, render departure of the 5′O less advanced compared with the solution reaction and stabilize charge buildup in the transition state. The ability to obtain a chemically detailed description of 2′-O-transphosphorylation transition states provides an opportunity to advance our understanding of biological catalysis significantly by determining how the catalytic modes and active site environments of phosphoryl transferases influence transition state structure. E nzymes achieve powerful rate enhancements by providing multiple catalytic modes to stabilize reaction transition states, including electrostatic interactions and proton transfer (1). For RNA 2′-O-transphosphorylation reactions, interactions with acid, base, or metal ion catalysts in solution can influence transition state structure (2). Understanding biological catalysis therefore requires knowledge of chemical mechanisms, transition state structure, and transition state interactions for both enzymatic and nonenzymatic RNA cleavage reactions. Comparisons of enzymatic and nonenzymatic catalysis can provide information on which catalytic modes are used by enzymes and whether unique features of the active site environment may alter the transition state charge distribution (3).RNA undergoes two competing transesterification reactions in solution: isomerization to a 2′,5′-phosphodiester and 2′-Otransphosphorylation to yield a cyclic 2′,3′-phosphodiester with concomitant release of the 5′O-nucleoside (4, 5). Reactions catalyzed by acid and by buffers yield both isomerization and cleavage products and proceed via a pentacoordinated phosphorane intermediate formed by an attack of the 2′OH at the adja...

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Section: Simulations and Qm Calculations To Model The Mechanism Andsupporting

confidence: 63%

“…Herschlag and colleagues (29,30) reported a large negative β LG , as well as a large leaving group KIE for both alkaline phosphatase (AP) and nonenzymatic monoester hydrolysis. These results are consistent with a dissociative transition state both on and off the enzyme.…”

Section: Simulations and Qm Calculations To Model The Mechanism Andmentioning

confidence: 99%

Experimental and computational analysis of the transition state for ribonuclease A-catalyzed RNA 2′- O -transphosphorylation

Gu¹,

Zhang²,

Wong

et al. 2013

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

125

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“…In addition to the different leaving group ability, the charge distribution on the leaving group is also significantly different. During phosphate ester hydrolysis, significant charge build-up occurs on the bridging oxygen of the leaving group and this charge is stabilized by the M1 ion (O’Brien and Herschlag, 2002, Zalatan, et al, 2007). For phosphonate hydrolysis, the charge is delocalized such that most of the charge likely resides on the oxygens of the acetate enolate (Figure 5A).…”

Section: Discussionmentioning

confidence: 99%

Structural and Mechanistic Insights into C-P Bond Hydrolysis by Phosphonoacetate Hydrolase

Agarwal

Borisova

Metcalf

et al. 2011

Chemistry & Biology

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SUMMARY Bacteria have evolved pathways to metabolize phosphonates as a nutrient source for phosphorus. In Sinorhizobium meliloti 1021, 2-aminoethylphosphonate is catabolized to phosphonoacetate, which is converted to acetate and inorganic phosphate by phosphonoacetate hydrolase (PhnA). Here we present detailed biochemical and structural characterization of PhnA that provides insights into the mechanism of C-P bond cleavage. The 1.35 Å resolution crystal structure reveals a catalytic core similar to those of alkaline phosphatases and nucleotide pyrophosphatases, but with notable differences such as a longer metal-metal distance. Detailed structure-guided analysis of active site residues and four additional co-crystal structures with phosphonoacetate substrate, acetate, phosphonoformate inhibitor, and a covalently-bound transition state mimic, provide insight into active site features that may facilitate cleavage of the C-P bond. These studies expand upon the array of reactions that can be catalyzed by enzymes of the alkaline phosphatase superfamily.

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“…In some cases, including for example carboxypeptidase A [34] and thermolysin [35], a single zinc ion possibly performs both of the above tasks, inducing the generation of both a nucleophilic and an electrophilic species that react with one another. In other cases, instead, two or three zinc ions are present in the active site of the enzyme and contribute to the catalytic mechanism, such as in alkaline phosphatase [36,37] and phospholipase C [38]. In fact, zinc hydrolases containing two or three catalytic zinc ions are not uncommon, representing together about 40% of zinc hydrolases with known structure.…”

Section: Roles Of Zinc In Zinc Enzymesmentioning

confidence: 99%

A bioinformatics view of zinc enzymes

Andreini

Bertini

2012

Journal of Inorganic Biochemistry

192

113

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Kinetic Isotope Effects for Alkaline Phosphatase Reactions: Implications for the Role of Active-Site Metal Ions in Catalysis

Cited by 68 publications

References 81 publications

Experimental and computational analysis of the transition state for ribonuclease A-catalyzed RNA 2′- O -transphosphorylation

Experimental and computational analysis of the transition state for ribonuclease A-catalyzed RNA 2′- O -transphosphorylation

Structural and Mechanistic Insights into C-P Bond Hydrolysis by Phosphonoacetate Hydrolase

A bioinformatics view of zinc enzymes

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