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. Conditions were established for efficient cross‐linking of recombinant bovine and yeast poly(A) polymerases to 8‐azido‐ATP. Mn 2+ strongly stimulated this reaction due to a 50‐fold lower K i for 8‐azido‐ATP in the presence of Mn 2+ . 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 Mn 2+ no longer stimulated the reaction. Sites of 8‐azido‐ATP crosslinking 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.