The Essential Functional Interplay of the Catalytic Groups in Acid Phosphatase



Cooperative interplay between the functional devices of a preorganized active site is fundamental to enzyme catalysis. A deepened understanding of this phenomenon is central to elucidating the remarkable efficiency of natural enzymes, and provides an essential benchmark for enzyme design and engineering. Here, we study the functional interconnectedness of the catalytic nucleophile (His18) in an acid phosphatase by analyzing the consequences of its replacement with aspartate. We present crystallographic, biochemical and computational evidence for a conserved mechanistic pathway via a phospho-enzyme intermediate on Asp18. Linear free-energy relationships for phosphoryl transfer from phosphomonoester substrates to His18/Asp18 provide evidence for cooperative interplay between the nucleophilic and general-acid catalytic groups in the wildtype enzyme, and its substantial loss in the H18D variant. As an isolated factor of phosphatase efficiency, the advantage of a histidine compared to an aspartate nucleophile is around 10^4-fold. Cooperativity with the catalytic acid adds ≥10^2-fold to that advantage. Empirical valence bond simulations of phosphoryl transfer from glucose 1-phosphate to His and Asp in the enzyme explain the loss of activity of the Asp18 enzyme through a combination of impaired substrate positioning in the Michaelis complex, as well as a shift from early to late protonation of the leaving group in the H18D variant. The evidence presented furthermore suggests that the cooperative nature of catalysis distinguishes the enzymatic reaction from the corresponding reaction in solution and is enabled by the electrostatic preorganization of the active site. Our results reveal sophisticated discrimination in multifunctional catalysis of a highly proficient phosphatase active site.


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Supplementary material

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Supporting Information
Supporting Information Preparation of ecAGP and auxiliary enzymes, Sections S1.2 to S1.7; assays used, Section S1.8; crystallization and structure determination, Section S1.9 -S1.10 and Table S9; kinetic studies and LFER analysis, sections S1.11-S1.12 and Table S14; isotope exchange studies, Section S1.14 and Figure S9; phospho-enzyme intermediate trapping studies, Sections S1.15 to S1.18, Figures S2 to S5, Table S3 to S6; preparation of aryl phosphates, Section S1.19, Figures S19 to S23; synthesis of (RP)- and (SP)-[16O,17O,18O]PEP, sections S1.20 to S1.27; stereochemical analysis of enzymatic phosphoryl transfer, sections S1.28 – S1.32, Figures S10 to S14, Tables S7 to S8; and EVB-calculations, Sections S1.33 to S1.36, Figures S15 to S16, Figures S24 to S26, and Tables S10, S13 and S15.

Supplementary weblinks

Crystal structure of Escherichia coli periplasmic glucose-1-phosphatase H18D mutant
X-tal structure of AGP H18D