Mechanism of phosphoanhydride cleavage by baculovirus phosphatase

Baculovirus phosphatase (BVP) is a member of the metazoan RNA triphosphatas e enzyme family that includes the RNA triphosphatase component of the mRNA capping apparatus, BVP and other metazoan RNA triphosphatases belong to a s uperfamily of phosphatases that act via the formation and hydrolysis of a c ovalent cysteinyl-phosphate intermediate. Here we demonstrate the formation of a BVP phosphoenzyme upon reaction with [gamma-P-32]ATP and identify the linkage as a thiophosphate based on its chemical lability, We surmise:that the phosphate is linked to Cys(119) of BVP because replacement of Cys(119) by alanine or serine abrogates phosphoenzyme formation and phosphohydrolas e activity. The catalytic cysteine is situated within a conserved phosphate -binding loop ((118)HCTHGINRTGY(128)). We show that all of the non-aliphati c side chains of the phosphate-binding loop are functionally important, ins ofar as mutants H118A, H121A, N124A, R125A, T126A, and Y128A were inactive in gamma phosphate hydrolysis and the T120A mutant was 7% as active as wild -type BVP, Structure-activity relationships at the essential positions: of the phosphate-binding loop were elucidated by conservative substitutions. A conserved aspartic acid (Asp(60)) invoked as a candidate general acid cata lyst was dispensable for phosphohydrolase activity and phosphoenzyme format ion by BVP, We propose that the low pK(a) of the bridging oxygen of the bet a phosphate leaving group circumvents a requirement for expulsion by a prot on donor during attack by cysteine on the gamma phosphorus. in contrast, a conserved aspartic acid is essential for the phosphomonoesterase reactions catalyzed by protein phosphatases, where the serine or tyrosine leaving gro ups have a much higher pK(a) than does ADP.