Transition-State Interactions in a Promiscuous Enzyme: Sulfate and Phosphate Monoester Hydrolysis by Pseudomonas aeruginosa Arylsulfatase.
Mohamed, Mark F
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van Loo, B., Berry, R., Boonyuen, U., Mohamed, M. F., Golicnik, M., Hengge, A. C., & Hollfelder, F. (2019). Transition-State Interactions in a Promiscuous Enzyme: Sulfate and Phosphate Monoester Hydrolysis by Pseudomonas aeruginosa Arylsulfatase.. Biochemistry, 58 (10), 1363-1378. https://doi.org/10.1021/acs.biochem.8b00996
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 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 (leaving groupobs-Enz=-0.33) than the uncatalyzed reaction (leaving groupobs=-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 (leaving 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 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).
Pseudomonas aeruginosa, Phosphates, Sulfates, Organophosphorus Compounds, Sulfatases, Arylsulfatases, Catalytic Domain, Substrate Specificity, Hydrolysis, Kinetics, Catalysis, Organophosphates
BBSRC BB/I004327/1 EPSRC EP/E019390/1)
External DOI: https://doi.org/10.1021/acs.biochem.8b00996
This record's URL: https://www.repository.cam.ac.uk/handle/1810/288717