Mechanism of phosphoanhydride cleavage by baculovirus phosphatase.
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 [gamma-(32)P]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 phosphohydrolase activity. The catalytic cysteine is situated within a conserved phosphate-binding loop ((118)HCTHGINRTGY(128)). 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 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 catalyst was dispensable for phosphohydrolase activity and phosphoenzyme formation by BVP. We propose that the low pK(a) of the bridging oxygen of the beta phosphate leaving group circumvents a requirement for expulsion by a proton 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 groups have a much higher pK(a) than does ADP.
Martins
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A
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
Shuman
S
S
D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D016367:Baculoviridae / Q000201:enzymology*; D002384:Catalysis; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D004252:DNA Mutational Analysis; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D010755:Organophosphates / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010758:Phosphorus / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D013329:Structure-Activity Relationship; D013696:Temperature; D013997:Time Factors; D014170:Transformation, Genetic
D010755:Organophosphates; D012333:RNA, Messenger; D011994:Recombinant Proteins; D010758:Phosphorus; D001224:Aspartic Acid; D012694:Serine; D000244:Adenosine Diphosphate; C417245:baculovirus phosphatase; D010744:Phosphoric Monoester Hydrolases; D000251:Adenosine Triphosphatases; D003545:Cysteine; D000409:Alanine; D010100:Oxygen