Related Experiment Videos
Mechanism of phosphoanhydride cleavage by baculovirus phosphatase
1Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
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 [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.
Insights
Baculovirus phosphatase (BVP) forms a covalent intermediate with ATP, identifying Cys119 as crucial for its RNA triphosphatase activity. This study elucidates key residues in BVP's active site, differentiating its mechanism from other phosphatases.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Baculovirus phosphatase (BVP) is an RNA triphosphatase enzyme.
- It belongs to a superfamily of phosphatases utilizing a cysteinyl-phosphate intermediate.
- This enzyme family is related to the mRNA capping apparatus.
Purpose of the Study:
- To investigate the catalytic mechanism of Baculovirus phosphatase (BVP).
- To identify the key residues involved in BVP's phosphohydrolase activity.
- To elucidate the structure-activity relationships within BVP's active site.
Main Methods:
- Demonstration of BVP phosphoenzyme formation using [gamma-(32)P]ATP.
- Chemical lability analysis to identify the phosphoenzyme linkage.
- Site-directed mutagenesis of BVP, including Cys119 substitutions and mutations in the phosphate-binding loop.
- Assays for phosphohydrolase activity and phosphoenzyme formation.
Main Results:
- BVP forms a labile thiophosphate-linked phosphoenzyme with ATP, indicating Cys119 is the catalytic residue.
- Mutations at Cys119 (C119A, C119S) abolish activity.
- All non-aliphatic residues in the conserved phosphate-binding loop (H118, H121, N124, R125, T126, Y128) are essential for activity.
- Mutant T120A shows significantly reduced activity.
- Conserved Asp60 is dispensable for BVP's phosphohydrolase activity.
Conclusions:
- Cys119 is the catalytic residue in BVP, forming a covalent cysteinyl-phosphate intermediate.
- The identified phosphate-binding loop residues are critical for enzyme function.
- BVP's mechanism may differ from other phosphatases due to the dispensability of Asp60 and the nature of the leaving group, potentially bypassing the need for a proton donor.