bi8b00996_si_001.pdf (1.51 MB)
Transition-State Interactions in a Promiscuous Enzyme: Sulfate and Phosphate Monoester Hydrolysis by Pseudomonas aeruginosa Arylsulfatase
journal contribution
posted on 2019-02-27, 16:19 authored by Bert van Loo, Ryan Berry, Usa Boonyuen, Mark F. Mohamed, Marko Golicnik, Alvan C. Hengge, Florian HollfelderPseudomonas aeruginosa arylsulfatase (PAS) hydrolyzes
sulfate and, promiscuously, phosphate monoesters. Enzyme-catalyzed
sulfate transfer is crucial to a wide variety of biological processes,
but detailed studies of the mechanistic contributions to its catalysis
are lacking. We present linear free energy relationships (LFERs) and
kinetic isotope effects (KIEs) of PAS and analyses of active site
mutants that suggest a key role for leaving group (LG) stabilization.
In LFERs PASWT has a much less negative Brønsted coefficient
(βleaving groupobs‑Enz = −0.33)
than the uncatalyzed reaction (βleaving groupobs = −1.81). This situation is diminished when
cationic active site groups are exchanged for alanine. The considerable
degree of bond breaking during the transition state (TS) is evidenced
by an 18Obridge KIE of 1.0088. LFER and KIE
data for several active site mutants point to leaving group stabilization
by active site K375, in cooperation with H211. 15N KIEs
and the increased sensitivity to leaving group ability of the sulfatase
activity in neat D2O (Δβleaving groupH‑D = +0.06) suggest that the mechanism for S–Obridge bond fission shifts, with decreasing leaving group ability,
from charge compensation via Lewis acid interactions toward direct
proton donation. 18Ononbridge KIEs indicate
that the TS for PAS-catalyzed sulfate monoester hydrolysis has a significantly
more associative character compared to the uncatalyzed reaction, while
PAS-catalyzed phosphate monoester hydrolysis does not show this shift.
This difference in enzyme-catalyzed TSs appears to be the major factor
favoring specificity toward sulfate over phosphate esters by this
promiscuous hydrolase, since other features are either too similar
(uncatalyzed TS) or inherently favor phosphate (charge).
History
Usage metrics
Categories
Keywords
chargebond18 O bridge KIEgroupPseudomonas aeruginosa Arylsulfatase Pseudomonas aeruginosa arylsulfataseobLFERs PAS WTstabilizationPhosphate Monoester HydrolysisPAS-catalyzed sulfate monoester hydrolysisEnzyme-catalyzed sulfate transfersite mutants pointLewis acid interactionsshiftuncatalyzedLGPAS-catalyzed phosphate monoester hydrolysisTSH 211. 15 N KIEsgroup ability18 O nonbridge KIEs
Licence
Exports
RefWorks
BibTeX
Ref. manager
Endnote
DataCite
NLM
DC