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Probing the origins of catalytic discrimination between phosphate and sulfate monoester hydrolysis: comparative analysis of alkaline phosphatase and protein tyrosine phosphatases.

Andrews LD, Zalatan JG, Herschlag D - Biochemistry (2014)

Bottom Line: 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 ∼10(10)-fold preference for phosphate monoester hydrolysis, despite the similarity between these reactions.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Systems Biology, ‡Department of Chemistry, and §Department of Biochemistry, Stanford University , Stanford, California 94305-5307, United States.

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 ∼10(10)-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 × 10(6) to 7 × 10(7), 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.

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Models of the electrostaticsurface potential for WT AP (A) andStp1 (B) with arrows pointing to the active site nucleophile. Formodeling AP, the X-ray structure from ref (57) was used (PDB code 3TG0). For modeling Stp1, the X-ray structureof low-molecular weight bovine PTP in ref (56) (PDB code 1Z12) was used to generate a structural homologymodel of Stp1 using the program Modeller.53 The protein surface is colored according to electrostatic potential(positive, blue; negative, red; ±6kT/e). For the electrostatic calculation, the active site nucleophilesof AP and Stp1 were deprotonated, and for Stp1, the Asp28 generalacid was protonated. Created with AMBER/ABPS in MacPyMOL.54,55
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fig4: Models of the electrostaticsurface potential for WT AP (A) andStp1 (B) with arrows pointing to the active site nucleophile. Formodeling AP, the X-ray structure from ref (57) was used (PDB code 3TG0). For modeling Stp1, the X-ray structureof low-molecular weight bovine PTP in ref (56) (PDB code 1Z12) was used to generate a structural homologymodel of Stp1 using the program Modeller.53 The protein surface is colored according to electrostatic potential(positive, blue; negative, red; ±6kT/e). For the electrostatic calculation, the active site nucleophilesof AP and Stp1 were deprotonated, and for Stp1, the Asp28 generalacid was protonated. Created with AMBER/ABPS in MacPyMOL.54,55

Mentions: Calculation of thesurface electrostatic potentials for AP and Stp1 using a Poisson–Boltzmannequation solver reveal positive potentials near the active sites (Figure 4) that could contribute to discrimination by preferentiallyattracting more negatively charged substrates into the active siteand thereby favor formation of the enzyme and substrate complex forthe more highly charged substrate. If a long-range attractive electrostaticpotential contributes to the AP and Stp1 catalyzed reactions, thepresence of counterions would be expected to decrease this attractionso that the catalytic activity would decrease as the ionic strengthof the reaction solution is raised and decrease more for a more highlycharged enzyme. Because increased ionic strength will screen long-rangecharge–charge interactions and not local interactions, thisexperiment cannot probe local electrostatic and dipolar interactionsthat are within the active site and involved in the progression fromthe enzyme/substrate to the enzyme/transition state complex. In addition,because nonideal effects of ions on enzymes are common, comparisonof the ionic strength dependence of reactions of the (phosphate ester)dianion versus (sulfate ester) monoanion substrates for each enzymeis important in controlling for such effects.


Probing the origins of catalytic discrimination between phosphate and sulfate monoester hydrolysis: comparative analysis of alkaline phosphatase and protein tyrosine phosphatases.

Andrews LD, Zalatan JG, Herschlag D - Biochemistry (2014)

Models of the electrostaticsurface potential for WT AP (A) andStp1 (B) with arrows pointing to the active site nucleophile. Formodeling AP, the X-ray structure from ref (57) was used (PDB code 3TG0). For modeling Stp1, the X-ray structureof low-molecular weight bovine PTP in ref (56) (PDB code 1Z12) was used to generate a structural homologymodel of Stp1 using the program Modeller.53 The protein surface is colored according to electrostatic potential(positive, blue; negative, red; ±6kT/e). For the electrostatic calculation, the active site nucleophilesof AP and Stp1 were deprotonated, and for Stp1, the Asp28 generalacid was protonated. Created with AMBER/ABPS in MacPyMOL.54,55
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222534&req=5

fig4: Models of the electrostaticsurface potential for WT AP (A) andStp1 (B) with arrows pointing to the active site nucleophile. Formodeling AP, the X-ray structure from ref (57) was used (PDB code 3TG0). For modeling Stp1, the X-ray structureof low-molecular weight bovine PTP in ref (56) (PDB code 1Z12) was used to generate a structural homologymodel of Stp1 using the program Modeller.53 The protein surface is colored according to electrostatic potential(positive, blue; negative, red; ±6kT/e). For the electrostatic calculation, the active site nucleophilesof AP and Stp1 were deprotonated, and for Stp1, the Asp28 generalacid was protonated. Created with AMBER/ABPS in MacPyMOL.54,55
Mentions: Calculation of thesurface electrostatic potentials for AP and Stp1 using a Poisson–Boltzmannequation solver reveal positive potentials near the active sites (Figure 4) that could contribute to discrimination by preferentiallyattracting more negatively charged substrates into the active siteand thereby favor formation of the enzyme and substrate complex forthe more highly charged substrate. If a long-range attractive electrostaticpotential contributes to the AP and Stp1 catalyzed reactions, thepresence of counterions would be expected to decrease this attractionso that the catalytic activity would decrease as the ionic strengthof the reaction solution is raised and decrease more for a more highlycharged enzyme. Because increased ionic strength will screen long-rangecharge–charge interactions and not local interactions, thisexperiment cannot probe local electrostatic and dipolar interactionsthat are within the active site and involved in the progression fromthe enzyme/substrate to the enzyme/transition state complex. In addition,because nonideal effects of ions on enzymes are common, comparisonof the ionic strength dependence of reactions of the (phosphate ester)dianion versus (sulfate ester) monoanion substrates for each enzymeis important in controlling for such effects.

Bottom Line: 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 ∼10(10)-fold preference for phosphate monoester hydrolysis, despite the similarity between these reactions.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Systems Biology, ‡Department of Chemistry, and §Department of Biochemistry, Stanford University , Stanford, California 94305-5307, United States.

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 ∼10(10)-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 × 10(6) to 7 × 10(7), 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.

Show MeSH
Related in: MedlinePlus