Experimental and Theoretical Insights into the Mechanisms of Sulfate and Sulfamate Ester Hydrolysis and the End Products of Type I Sulfatase Inactivation by Aryl Sulfamates

Abstract

Type I sulfatases catalyze the hydrolysis of sulfate esters through S-O bond cleavage and possess a catalytically essential formylglycine (FGly) active-site residue that is post-translationally derived from either cysteine or serine. Type I sulfatases are inactivated by aryl sulfamates in a time-dependent, irreversible, and active-site directed manner consistent with covalent modification of the active site. We report a theoretical (SCS-MP2//B3LYP) and experimental study of the uncatalyzed and enzyme-catalyzed hydrolysis of aryl sulfates and sulfamates. In solution, aryl sulfate monoanions undergo hydrolysis by an S(N)2 mechanism whereas aryl sulfamate monoanions follow an S(N)1 pathway with SO2NH as an intermediate; theory traces this difference to the markedly greater stability of SO2NH versus SO3. For Pseudomonas aeruginosa arylsulfatase-catalyzed aryl sulfate hydrolysis, Brønsted analysis (log(V(max)/K(M)) versus leaving group pK(a) value) reveals β(LG) = -0.86 ± 0.23, consistent with an S(N)2 at sulfur reaction but substantially smaller than that reported for uncatalyzed hydrolysis (β(LG) = -1.81). Common to all proposed mechanisms of sulfatase catalysis is a sulfated FGly intermediate. Theory indicates a ≥26 kcal/mol preference for the intermediate to release HSO4(-) by an E2 mechanism, rather than alkaline phosphatase-like S(N)2 substitution by water. An evaluation of the stabilities of various proposed end-products of sulfamate-induced sulfatase inactivation highlights that an imine N-sulfate derived from FGly is the most likely irreversible adduct.