Essential Functional Interplay of the Catalytic Groups in Acid Phosphatase
Pfeiffer, Martin ; Crean, Rory M ; Moreira, Catia ; Parracino, Antonietta ; Oberdorfer, Gustav ; Brecker, Lothar ; Hammerschmidt, Friedrich ; Kamerlin, Shina Caroline Lynn LU
and Nidetzky, Bernd
(2022)
In ACS Catalysis
12(6).
p.3357-3370
- Abstract
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... (More)
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.
(Less)
- author
- Pfeiffer, Martin
; Crean, Rory M
; Moreira, Catia
; Parracino, Antonietta
; Oberdorfer, Gustav
; Brecker, Lothar
; Hammerschmidt, Friedrich
; Kamerlin, Shina Caroline Lynn
LU
and Nidetzky, Bernd
- publishing date
- 2022-03-18
- type
- Contribution to journal
- publication status
- published
- in
- ACS Catalysis
- volume
- 12
- issue
- 6
- pages
- 14 pages
- publisher
- The American Chemical Society (ACS)
- external identifiers
-
- scopus:85126083995
- pmid:35356705
- ISSN
- 2155-5435
- DOI
- 10.1021/acscatal.1c05656
- language
- English
- LU publication?
- no
- additional info
- © 2022 The Authors. Published by American Chemical Society.
- id
- 6089dd9b-ee4a-4918-9b72-998bee88a5ff
- date added to LUP
- 2025-01-11 18:47:43
- date last changed
- 2025-07-13 18:41:13
@article{6089dd9b-ee4a-4918-9b72-998bee88a5ff,
abstract = {{<p>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.</p>}},
author = {{Pfeiffer, Martin and Crean, Rory M and Moreira, Catia and Parracino, Antonietta and Oberdorfer, Gustav and Brecker, Lothar and Hammerschmidt, Friedrich and Kamerlin, Shina Caroline Lynn and Nidetzky, Bernd}},
issn = {{2155-5435}},
language = {{eng}},
month = {{03}},
number = {{6}},
pages = {{3357--3370}},
publisher = {{The American Chemical Society (ACS)}},
series = {{ACS Catalysis}},
title = {{Essential Functional Interplay of the Catalytic Groups in Acid Phosphatase}},
url = {{http://dx.doi.org/10.1021/acscatal.1c05656}},
doi = {{10.1021/acscatal.1c05656}},
volume = {{12}},
year = {{2022}},
}