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J. Biol. Chem., Vol. 275, Issue 45, 35070-35076, November 10, 2000

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Mechanism of Phosphoanhydride Cleavage by Baculovirus Phosphatase^*

Alexandra Martins and Stewart Shuman

From the Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021

Baculovirus phosphatase (BVP) is a member of the metazoan RNA triphosphatase enzyme family that includes the RNA triphosphatase component of the mRNA capping apparatus. BVP and other metazoan RNA triphosphatases belong to a superfamily of phosphatases that act via the formation and hydrolysis of a covalent cysteinyl-phosphate intermediate. Here we demonstrate the formation of a BVP phosphoenzyme upon reaction with [-³²P]ATP and identify the linkage as a thiophosphate based on its chemical lability. We surmise that the phosphate is linked to Cys¹¹⁹ of BVP because replacement of Cys¹¹⁹ by alanine or serine abrogates phosphoenzyme formation and phosphohydrolase activity. The catalytic cysteine is situated within a conserved phosphate-binding loop (¹¹⁸HCTHGINRTGY¹²⁸). We show that all of the non-aliphatic side chains of the phosphate-binding loop are functionally important, insofar as mutants H118A, H121A, N124A, R125A, T126A, and Y128A were inactive in  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 (Asp⁶⁰) invoked as a candidate general acid catalyst was dispensable for phosphohydrolase activity and phosphoenzyme formation by BVP. We propose that the low pK_a of the bridging oxygen of the  phosphate leaving group circumvents a requirement for expulsion by a proton donor during attack by cysteine on the  phosphorus. In contrast, a conserved aspartic acid is essential for the phosphomonoesterase reactions catalyzed by protein phosphatases, where the serine or tyrosine leaving groups have a much higher pK_a than does ADP.

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