Functional groups required for the stability of yeast RNA triphosphatase in vitro and in vivo Journal Article
| Authors: | Bisaillon, M.; Shuman, S. |
| Article Title: | Functional groups required for the stability of yeast RNA triphosphatase in vitro and in vivo |
| Abstract: | Cet1, the RNA triphosphatase component of the yeast mRNA capping apparatus, catalyzes metal-dependent γ-phosphate hydrolysis within the hydrophilic interior of an eight-strand β barrel (the "triphosphate tunnel"), which rests upon a globular protein core (the "pedestal"). We performed a structure-guided alanine scan of 17 residues located in the tunnel (Ser373, Thr375, Gln405, His411, Ser429, Glu488, Thr490), on the tunnel's outer surface (Ser378, Ser487, Thr489, His 491), at the tunnel-pedestal interface (Ile304, Met 308) and in the pedestal (Asp315, Lys317, Arg321, Asp425). Alanine mutations at 14 positions had no significant effect on Cet1 phosphohydrolase activity in vitro and had no effect on Cet1 function in vivo. Two of the mutations (R321A and D425A) elicited a thermosensitive (ts) yeast growth phenotype. The R321A and D425A proteins had full phosphohydrolase activity in vitro, but were profoundly thermolabile. Arg321 and Asp425 interact to form a salt bridge within the pedestal that tethers two of the strands of the tunnel. Mutations R321Q and D411N resulted in ts defects in vivo and in vitro, as did the double-mutant R321A-D435A, whereas the R321K protein was fully stable in vivo and in vitro. These results highlight the critical role of the buried salt bridge in Cet1 stability. Replacement of Ser429 by alanine or valine elicited a cold-sensitive (cs) yeast growth phenotype. The S429A and S429V proteins were fully active when produced in bacteria at 37°C, but were inactive when produced at 17°C. Replacement of Ser429 by threonine partially suppressed the cold sensitivity of the Cet1 phosphohydrolase, but did not suppress the cs growth defect in yeast. |
| Keywords: | unclassified drug; mutation; nonhuman; molecular genetics; mutant protein; proteins; phenotype; metabolism; cell division; amino acid substitution; phosphatase; serine; protein binding; enzymology; acid anhydride hydrolase; rna triphosphatase; enzyme activity; acid anhydride hydrolases; chemistry; amino acid sequence; conserved sequence; molecular sequence data; sequence homology, amino acid; saccharomyces cerevisiae; nucleotide sequence; temperature; amino acid; models, molecular; isoleucine; threonine; chemical structure; yeast; alanine; amino acids; protein folding; sequence homology; adenosine triphosphatase; biochemistry; glutamic acid; catalytic domain; phosphate; phosphates; adenosine triphosphatases; hydrolysis; aspartic acid; mutagenesis; glutamine; methionine; lysine; arginine; histidine; enzyme active site; hydrophilicity; rna capping; enzyme stability; bacteriology; cold sensitivity; protein cet1; growth kinetics; priority journal; article; globular protein; thermosensitivity |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 276 |
| Issue: | 32 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 2001-08-10 |
| Start Page: | 30514 |
| End Page: | 30520 |
| Language: | English |
| DOI: | 10.1074/jbc.M104936200 |
| PUBMED: | 11395522 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Export Date: 21 May 2015 -- Source: Scopus |
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