Probing the Origins of Catalytic Discrimination between Phosphate and Sulfate Monoester Hydrolysis: Comparative Analysis of Alkaline Phosphatase and Protein Tyrosine Phosphatases

Andrews, Logan D.; Zalatan, Jesse G.; Herschlag, Daniel

doi:10.1021/bi500765p

Cited by 28 publications

(38 citation statements)

References 55 publications

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“…Along each P–O bond, bond lengths and charge separations increase somewhat upon passage from the ground state to the transition state (134). This gives AP three “sites” at which to perform electric field catalysis.…”

Section: Unifying Consequences Of the Electric Field Catalysis Modelmentioning

confidence: 99%

“…One of the best case studies for enzyme promiscuity is AP, which in addition to its primary function of hydrolyzing phosphate monoesters, possesses sulfatase activity (134, 144). The structure and charge pattern of the transition states for these two reactions are nearly identical (Figure 6 c ), but the magnitude of the charge on each of the three nonbridging oxygen atoms is approximately half that of an analogous phosphate.…”

Section: Unifying Consequences Of the Electric Field Catalysis Modelmentioning

confidence: 99%

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Electric Fields and Enzyme Catalysis

Fried

Boxer

2017

Annu. Rev. Biochem.

385

456

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What happens inside an enzyme’s active site to allow slow and difficult chemical reactions to occur so rapidly? This question has occupied biochemists’ attention for a long time. Computer models of increasing sophistication have predicted an important role for electrostatic interactions in enzymatic reactions, yet this hypothesis has proved vexingly difficult to test experimentally. Recent experiments utilizing the vibrational Stark effect make it possible to measure the electric field a substrate molecule experiences when bound inside its enzyme’s active site. These experiments have provided compelling evidence supporting a major electrostatic contribution to enzymatic catalysis. Here, we review these results and develop a simple model for electrostatic catalysis that enables us to incorporate disparate concepts introduced by many investigators to describe how enzymes work into a more unified framework stressing the importance of electric fields at the active site.

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Section: Unifying Consequences Of the Electric Field Catalysis Modelmentioning

confidence: 99%

Section: Unifying Consequences Of the Electric Field Catalysis Modelmentioning

confidence: 99%

Electric Fields and Enzyme Catalysis

Fried

Boxer

2017

Annu. Rev. Biochem.

385

456

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“…Members of the alkaline phosphatase (AP) superfamily constitute paradigmatic examples to study how new functions-in particular with seemingly contrasting or even incompatible catalytic requirements-can emerge in one scaffold (32)(33)(34)(35)(36). The AP superfamily represents a large cluster of enzymes that catalyze a broad range of chemically distinct reactions, including the hydrolysis of sulfate, phosphate, and phosphonate monoesters or phosphodiesters (37) (Fig.…”

Section: Significancementioning

confidence: 99%

Evolutionary repurposing of a sulfatase: A new Michaelis complex leads to efficient transition state charge offset

Miton

Jonas

Fischer

et al. 2018

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

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The recruitment and evolutionary optimization of promiscuous enzymes is key to the rapid adaptation of organisms to changing environments. Our understanding of the precise mechanisms underlying enzyme repurposing is, however, limited: What are the active-site features that enable the molecular recognition of multiple substrates with contrasting catalytic requirements? To gain insights into the molecular determinants of adaptation in promiscuous enzymes, we performed the laboratory evolution of an arylsulfatase to improve its initially weak phenylphosphonate hydrolase activity. The evolutionary trajectory led to a 100,000-fold enhancement of phenylphosphonate hydrolysis, while the native sulfate and promiscuous phosphate mono- and diester hydrolyses were only marginally affected (≤50-fold). Structural, kinetic, and in silico characterizations of the evolutionary intermediates revealed that two key mutations, T50A and M72V, locally reshaped the active site, improving access to the catalytic machinery for the phosphonate. Measured transition state (TS) charge changes along the trajectory suggest the creation of a new Michaelis complex (E•S, enzyme-substrate), with enhanced leaving group stabilization in the TS for the promiscuous phosphonate ( from -1.08 to -0.42). Rather than altering the catalytic machinery, evolutionary repurposing was achieved by fine-tuning the molecular recognition of the phosphonate in the Michaelis complex, and by extension, also in the TS. This molecular scenario constitutes a mechanistic alternative to adaptation solely based on enzyme flexibility and conformational selection. Instead, rapid functional transitions between distinct chemical reactions rely on the high reactivity of permissive active-site architectures that allow multiple substrate binding modes.

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“…Multi-faceted comparisons of structure, sequence, and cognate and promiscuous reactions of these superfamily members and their mutants in this work and previously have extended our mechanistic understanding, have led to models for the roles of active site and more distal structural elements, and have suggested evolutionary driving forces and adaptations. 17,24,32,34−36,39,52,53,57,59,60,88,96−99 …”

Section: General Implicationsmentioning

confidence: 99%

Mechanistic and Evolutionary Insights from Comparative Enzymology of Phosphomonoesterases and Phosphodiesterases across the Alkaline Phosphatase Superfamily

et al. 2016

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Naively one might have expected an early division between phosphate monoesterases and diesterases of the alkaline phosphatase (AP) superfamily. On the contrary, prior results and our structural and biochemical analyses of phosphate monoesterase PafA, from Chryseobacterium meningosepticum, indicate similarities to a superfamily phosphate diesterase [Xanthomonas citri nucleotide pyrophosphatase/phosphodiesterase (NPP)] and distinct differences from the three metal ion AP superfamily monoesterase, from Escherichia coli AP (EcAP). We carried out a series of experiments to map out and learn from the differences and similarities between these enzymes. First, we asked why there would be independent instances of monoesterases in the AP superfamily? PafA has a much weaker product inhibition and slightly higher activity relative to EcAP, suggesting that different metabolic evolutionary pressures favored distinct active-site architectures. Next, we addressed the preferential phosphate monoester and diester catalysis of PafA and NPP, respectively. We asked whether the >80% sequence differences throughout these scaffolds provide functional specialization for each enzyme’s cognate reaction. In contrast to expectations from this model, PafA and NPP mutants with the common subset of active-site groups embedded in each native scaffold had the same monoesterase:diesterase specificities; thus, the >107-fold difference in native specificities appears to arise from distinct interactions at a single phosphoryl substituent. We also uncovered striking mechanistic similarities between the PafA and EcAP monoesterases, including evidence for ground-state destabilization and functional active-site networks that involve different active-site groups but may play analogous catalytic roles. Discovering common network functions may reveal active-site architectural connections that are critical for function, and identifying regions of functional modularity may facilitate the design of new enzymes from existing promiscuous templates. More generally, comparative enzymology and analysis of catalytic promiscuity can provide mechanistic and evolutionary insights.

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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

Cited by 28 publications

References 55 publications

Electric Fields and Enzyme Catalysis

Electric Fields and Enzyme Catalysis

Evolutionary repurposing of a sulfatase: A new Michaelis complex leads to efficient transition state charge offset

Mechanistic and Evolutionary Insights from Comparative Enzymology of Phosphomonoesterases and Phosphodiesterases across the Alkaline Phosphatase Superfamily

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