Do Electrostatic Interactions with Positively Charged Active Site Groups Tighten the Transition State for Enzymatic Phosphoryl Transfer?

• Published in: Journal of the American Chemical Society Vol. 126; no. 38; pp. 11814 - 11819
• Main Authors: Nikolic-Hughes, Ivana, Rees, Douglas C, Herschlag, Daniel
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 29.09.2004
• Subjects: Alkaline Phosphatase - chemistry; Alkaline Phosphatase - metabolism; Binding Sites; Biological and medical sciences; Fundamental and applied biological sciences. Psychology; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Linear Energy Transfer; Mechanisms. Catalysis. Electron transfer. Models; Models, Molecular; Molecular biophysics; Organophosphates - chemistry; Organophosphates - metabolism; Physical chemistry in biology; Static Electricity; Sulfuric Acid Esters - chemistry; Sulfuric Acid Esters - metabolism; Alkaline phosphatase; Catalytic reaction; Enzyme; Electrostatic interaction; Phosphoric monoester hydrolases; Enzymatic catalysis; Molecular interaction; Esterases; Organic thiophosphate; Hydrolysis; Organic solvent; Organic sulfate; Reaction mechanism; Hydrolases; Kinetic parameter; Rate constant; Catalyst activity; Ultraviolet visible spectrometry
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja0480421

The effect of electrostatic interactions on the transition-state character for enzymatic phosphoryl transfer has been a subject of much debate. In this work, we investigate the transition state for alkaline phosphatase (AP) using linear free-energy relationships (LFERs). We determined k cat/K M for a series of aryl sulfate ester monoanions to obtain the Brønsted coefficient, βlg, and compared the value to that obtained previously for a series of aryl phosphorothioate ester dianion substrates. Despite the difference in substrate charge, the observed Brønsted coefficients for AP-catalyzed aryl sulfate and aryl phosphorothioate hydrolysis (−0.76 ± 0.14 and −0.77 ± 0.10, respectively) are strikingly similar, with steric effects being responsible for the uncertainties in these values. Aryl sulfates and aryl phosphates react via similar loose transition states in solution. These observations suggest an apparent equivalency of the transition states for phosphorothioate and sulfate hydrolysis reactions at the AP active site and, thus, negligible effects of active site electrostatic interactions on charge distribution in the transition state.