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Mapping of ATP binding regions in poly(A) polymerases by photoaffinity labeling and by mutational analysis identifies a domain conserved in many nucleotidyltransferases

Martin, G. and Jenö, P. and Keller, W.. (1999) Mapping of ATP binding regions in poly(A) polymerases by photoaffinity labeling and by mutational analysis identifies a domain conserved in many nucleotidyltransferases. Protein Science, 8 (11). pp. 2380-2391.

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Abstract

We have identified regions in poly(A) polymerases that interact with ATP. Conditions were established for efficient cross-linking of recombinant bovine and yeast poly(A) polymerases to 8-azido-ATP. Mn2+ strongly stimulated this reaction due to a 50-fold lower Ki for 8-azido-ATP in the presence of Mn2+. Mutations of the highly conserved Asp residues 113, 115, and 167, critical for metal binding in the catalytic domain of bovine poly(A) polymerase, led to a strong reduction of cross-linking efficiency, and Mn2+ no longer stimulated the reaction. Sites of 8-azido-ATP cross-linking were mapped in different poly(A) polymerases by CNBr-cleavage and analysis of tryptic peptides by mass spectroscopy. The main cross-link in Schizosaccharomyces pombe poly(A) polymerase could be assigned to the peptide DLELSDNNLLK (amino acids 167-177). Database searches with sequences surrounding the cross-link site detected significant homologies to other nucleotidyltransferase families, suggesting a conservation of the nucleotide-binding fold among these families of enzymes. Mutations in the region of the "helical turn motif" (a domain binding the triphosphate moiety of the nucleotide) and in the suspected nucleotide-binding helix of bovine poly(A) polymerase impaired ATP binding and catalysis. The results indicate that ATP is bound in part by the helical turn motif and in part by a region that may be a structural analog to the fingers domain found in many polymerases.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Former Organization Units Biozentrum > Cell Biology (Keller)
05 Faculty of Science > Departement Biozentrum > Growth & Development
05 Faculty of Science > Departement Biozentrum > Services Biozentrum > Mass Spectrometry (Jenö)
UniBasel Contributors:Keller, Walter and Jenö, Paul
Item Type:Article, refereed
Bibsysno:Link to catalogue
Publisher:Cambridge University Press
ISSN:0961-8368
e-ISSN:1469-896X
Note:Publication type according to Uni Basel Research Database: Journal article
Language:English
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Last Modified:08 Nov 2017 07:31
Deposited On:22 Mar 2012 13:29

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