Crystal structure of an enzyme-substrate complex provides insight into theinteraction between human arylsulfatase A and its substrates during catalysis

Arylsulfatase A (ASA) belongs to the sulfatase family whose members carry a C-alpha-formylglycine that is post-translationally generated by oxidation of a conserved cysteine or serine residue. The crystal structures of two ar ylsulfatases, ASA and ASB, and kinetic studies on ASA mutants led to differ ent proposals for the catalytic mechanism in the hydrolysis of sulfate este rs. The structures of two ASA mutants that lack the functional C-alpha-formylgl ycine residue 69, in complex with a synthetic substrate, have been determin ed in order to unravel the reaction mechanism. The crystal structure of the inactive mutant C69A-ASA in complex with p-nitrocatechol sulfate (pNCS) mi mics a reaction intermediate during sulfate ester hydrolysis by the active enzyme, without the covalent bond to the key side-chain FGly69. The structu re shows that the side-chains of lysine 123, lysine 302, serine 150, histid ine 229, the main-chain of the key residue 69 and the divalent cation in th e active center are involved in sulfate binding. It is proposed that histid ine 229 protonates the leaving alcoholate after hydrolysis. C69S-ASA is able to bind covalently to the substrate and hydrolyze it, but is unable to release the resulting sulfate. Nevertheless, the resulting sul fation is low. The structure of C69S-ASA shows the serine side-chain in a s ingle conformation, turned away from the position a substrate occupies in t he complex. This suggests that the double conformation observed in the stru cture of wild-tips ASA is more likely to correspond to a formylglycine hydr ate than to a twofold disordered aldehyde oxo group, and accounts for the r elative inertness of the C69S-ASA mutant. In the C69S-ASA-pNCS complex, the substrate occupies the same position as in the C69A-ASA-pNCS complex, whic h corresponds to the noncovalently bonded substrate. Based on the structura l data, a detailed mechanism for sulfate ester cleavage is proposed, involv ing an aldehyde hydrate as the functional group. (C) 2001 Academic Press.