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

We have identified regions in poly(A) polymerases that interact with ATP. C onditions were established for efficient cross-linking of recombinant bovin e and yeast poly(A) polymerases to 8-azido-ATP. Mn2+ strongly stimulated th is reaction due to a 50-fold lower K-i for 8-azido-ATP in the presence of M n2+. Mutations of the highly conserved Asp residues 113. 115, and 167, crit ical 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 pep tides by mass spectroscopy. The main cross-link in Schizosaccharomyces pomb e poly(A) polymerase could be assigned to the peptide DLELSDNNLLK (amino ac ids 167-177). Database searches with sequences surrounding the cross-link s ite detected significant homologies to other nucleotidyltransferase familie s, suggesting a conservation of the nucleotide-binding fold among these fam ilies of enzymes. Mutations in the region of the "helical rum motif" (a dom ain 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 helic al turn motif and in part by a region that may be a structural analog to th e fingers domain found in many polymerases.