TY - JOUR
T1 - Essential Functional Interplay of the Catalytic Groups in Acid Phosphatase
AU - Pfeiffer, Martin
AU - Crean, Rory M
AU - Moreira, Catia
AU - Parracino, Antonietta
AU - Oberdorfer, Gustav
AU - Brecker, Lothar
AU - Hammerschmidt, Friedrich
AU - Kamerlin, Shina Caroline Lynn
AU - Nidetzky, Bernd
N1 - Publisher Copyright:
© 2022 The Authors. Published by American Chemical Society.
PY - 2022
Y1 - 2022
N2 - The cooperative interplay between the functional devices of a preorganized active site is fundamental to enzyme catalysis. An in-depth 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 the cooperative interplay between the nucleophilic and general-acid catalytic groups in the wild-type 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 ∼104-fold. Cooperativity with the catalytic acid adds ≥102-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.
AB - The cooperative interplay between the functional devices of a preorganized active site is fundamental to enzyme catalysis. An in-depth 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 the cooperative interplay between the nucleophilic and general-acid catalytic groups in the wild-type 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 ∼104-fold. Cooperativity with the catalytic acid adds ≥102-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.
KW - enzyme catalysis
KW - EVB simulations
KW - functional cooperativity
KW - linear free-energy relationship
KW - nucleophilic catalysis
KW - phosphate transfer
UR - http://www.scopus.com/inward/record.url?scp=85126083995&partnerID=8YFLogxK
U2 - 10.1021/acscatal.1c05656
DO - 10.1021/acscatal.1c05656
M3 - Article
C2 - 35356705
AN - SCOPUS:85126083995
SN - 2155-5435
VL - 12
SP - 3357
EP - 3370
JO - ACS Catalysis
JF - ACS Catalysis
IS - 6
ER -