Mechanism of phosphoanhydride cleavage by baculovirus phosphatase Journal Article
| Authors: | Martins, A.; Shuman, S. |
| Article Title: | Mechanism of phosphoanhydride cleavage by baculovirus phosphatase |
| Abstract: | 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 [γ-32P]ATP and identify the linkage as a thiophosphate based on its chemical lability. We surmise thai 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 γ 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 β phosphate leaving group circumvents a requirement for expulsion by a proton donor during attack by cysteine on the γ 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. |
| Keywords: | mutation, missense; nonhuman; animals; phosphatase; serine; oxygen; dose-response relationship, drug; adenosine diphosphate; enzyme activity; structure-activity relationship; time factors; cloning, molecular; amino acid sequence; molecular sequence data; sequence homology, amino acid; kinetics; rna, messenger; escherichia coli; recombinant proteins; plasmids; temperature; mutagenesis, site-directed; dna mutational analysis; alanine; catalysis; enzyme binding; adenosine triphosphatases; hydrolysis; phosphoric monoester hydrolases; aspartic acid; catalyst; reaction analysis; cysteine; hydrogen-ion concentration; enzyme synthesis; phosphoric acid esters; metazoa; phosphorus; enzyme conformation; transformation, genetic; baculovirus; baculoviridae; unidentified baculovirus; humans; human; priority journal; article |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 275 |
| Issue: | 45 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 2000-11-10 |
| Start Page: | 35070 |
| End Page: | 35076 |
| Language: | English |
| DOI: | 10.1074/jbc.M005748200 |
| PUBMED: | 10954717 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Export Date: 18 November 2015 -- Source: Scopus |
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