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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: 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.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.

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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Analysis ofrelative rate enhancements for cognate reactions withtheir ability to discriminate between phosphate and sulfate estersubstrates. (A) Correlation of the enzymatic rate enhancement of pNPP hydrolysis with the extent of catalytic discriminationbetween pNPP and pNPS hydrolysisby AP superfamily members and PTPs. Data are from results herein andprior studies and are summarized in Table S1, Supporting Information. The linear least-squares fit has aslope of 0.9 log units (R2 = 0.74). Key:circles for AP and mutants thereof; diamond for PafA; squares forPTPs; triangle for PAc; inverted triangle for NPP; right angle trianglefor PMH; open circle for PAS. See Table S1, SupportingInformation, for enzyme abbreviations. The data point for PASwas not included in the fit because this enzyme prefers to hydrolyze pNPS over pNPP. (B) Plot of the enzymaticrate enhancement of pNPP hydrolysis with the enzymaticrate enhancement of pNPS hydrolysis by AP superfamilymembers and PTPs (excluding the AP superfamily member PAS). Key: sameas in panel A). The data point for PAS is omitted to allow bettervisualization of the other data points but is included in an analogousplot in the Supporting Information (FigureS8).
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fig6: Analysis ofrelative rate enhancements for cognate reactions withtheir ability to discriminate between phosphate and sulfate estersubstrates. (A) Correlation of the enzymatic rate enhancement of pNPP hydrolysis with the extent of catalytic discriminationbetween pNPP and pNPS hydrolysisby AP superfamily members and PTPs. Data are from results herein andprior studies and are summarized in Table S1, Supporting Information. The linear least-squares fit has aslope of 0.9 log units (R2 = 0.74). Key:circles for AP and mutants thereof; diamond for PafA; squares forPTPs; triangle for PAc; inverted triangle for NPP; right angle trianglefor PMH; open circle for PAS. See Table S1, SupportingInformation, for enzyme abbreviations. The data point for PASwas not included in the fit because this enzyme prefers to hydrolyze pNPS over pNPP. (B) Plot of the enzymaticrate enhancement of pNPP hydrolysis with the enzymaticrate enhancement of pNPS hydrolysis by AP superfamilymembers and PTPs (excluding the AP superfamily member PAS). Key: sameas in panel A). The data point for PAS is omitted to allow bettervisualization of the other data points but is included in an analogousplot in the Supporting Information (FigureS8).

Mentions: Figure 6A showsthe phosphate/sulfate monoesterdiscrimination as a function of the catalytic efficiency for hydrolysisof phosphate monoester dianions for all enzymes for which these parametershave been measured of which we are aware. Based on this limited sampleset, there is a strong correlation, but it appears that sulfataseactivity remains at a similar level (Figure 6B) so that the correlation is driven by increased phosphatase activity;that is, as phosphatase activity is increased, sulfatase activitydoes not increase commensurately (Figure 6B).Active site features that provide optimal phosphatase catalysis mayaffect only or predominantly this reaction, or increased catalyticpower toward phosphate esters may be accompanied by selective pressureagainst also increasing activity for sulfatase side reactions.


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)

Analysis ofrelative rate enhancements for cognate reactions withtheir ability to discriminate between phosphate and sulfate estersubstrates. (A) Correlation of the enzymatic rate enhancement of pNPP hydrolysis with the extent of catalytic discriminationbetween pNPP and pNPS hydrolysisby AP superfamily members and PTPs. Data are from results herein andprior studies and are summarized in Table S1, Supporting Information. The linear least-squares fit has aslope of 0.9 log units (R2 = 0.74). Key:circles for AP and mutants thereof; diamond for PafA; squares forPTPs; triangle for PAc; inverted triangle for NPP; right angle trianglefor PMH; open circle for PAS. See Table S1, SupportingInformation, for enzyme abbreviations. The data point for PASwas not included in the fit because this enzyme prefers to hydrolyze pNPS over pNPP. (B) Plot of the enzymaticrate enhancement of pNPP hydrolysis with the enzymaticrate enhancement of pNPS hydrolysis by AP superfamilymembers and PTPs (excluding the AP superfamily member PAS). Key: sameas in panel A). The data point for PAS is omitted to allow bettervisualization of the other data points but is included in an analogousplot in the Supporting Information (FigureS8).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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fig6: Analysis ofrelative rate enhancements for cognate reactions withtheir ability to discriminate between phosphate and sulfate estersubstrates. (A) Correlation of the enzymatic rate enhancement of pNPP hydrolysis with the extent of catalytic discriminationbetween pNPP and pNPS hydrolysisby AP superfamily members and PTPs. Data are from results herein andprior studies and are summarized in Table S1, Supporting Information. The linear least-squares fit has aslope of 0.9 log units (R2 = 0.74). Key:circles for AP and mutants thereof; diamond for PafA; squares forPTPs; triangle for PAc; inverted triangle for NPP; right angle trianglefor PMH; open circle for PAS. See Table S1, SupportingInformation, for enzyme abbreviations. The data point for PASwas not included in the fit because this enzyme prefers to hydrolyze pNPS over pNPP. (B) Plot of the enzymaticrate enhancement of pNPP hydrolysis with the enzymaticrate enhancement of pNPS hydrolysis by AP superfamilymembers and PTPs (excluding the AP superfamily member PAS). Key: sameas in panel A). The data point for PAS is omitted to allow bettervisualization of the other data points but is included in an analogousplot in the Supporting Information (FigureS8).
Mentions: Figure 6A showsthe phosphate/sulfate monoesterdiscrimination as a function of the catalytic efficiency for hydrolysisof phosphate monoester dianions for all enzymes for which these parametershave been measured of which we are aware. Based on this limited sampleset, there is a strong correlation, but it appears that sulfataseactivity remains at a similar level (Figure 6B) so that the correlation is driven by increased phosphatase activity;that is, as phosphatase activity is increased, sulfatase activitydoes not increase commensurately (Figure 6B).Active site features that provide optimal phosphatase catalysis mayaffect only or predominantly this reaction, or increased catalyticpower toward phosphate esters may be accompanied by selective pressureagainst also increasing activity for sulfatase side reactions.

Bottom Line: 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.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.

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