Document Type

Article

Journal/Book Title

Biochemistry

Publication Date

2-27-2019

Publisher

American Chemical Society

Volume

58

Issue

10

First Page

1

Last Page

55

Abstract

Pseudomonas aeruginosa arylsulfatase (PAS) hydrolyses 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 active site mutants that suggest a key role for leaving group (LG) stabilization. In LFERs PASWT has a much less negative Brønsted coefficient (ßleaving group obs-Enz=-0.33) than the uncatalyzed reaction (ßleaving group obs=-1.81). This situation is diminished when cationic active site groups are exchanged for alanine. The considerable degree of bond breaking during the 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 ([delta]ßleaving group H-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 towards 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).

Comments

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Biochemistry, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://pubs.acs.org/doi/10.1021/acs.biochem.8b00996

Available for download on Thursday, February 27, 2020

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