Roles of N-terminal pyroglutamate in maintaining structural integrity and pKa values of catalytic histidine residues in bullfrog ribonuclease 3

Many proteins and bioactive peptides contain an N-terminal pyroglutamate residue (Pyr1). This residue reduces the susceptibility of the protein to aminopeptidases and often has important functional roles. The antitumor ribonuclease RC-RNase 3 (RNase 3) from oocytes of Rana catesbeiana (bullfrog) is one such protein. We have produced recombinant RNase 3 containing the N-terminal Pyr1 (pRNase 3) and found it to be indistinguishable from the native RNase 3 by mass spectrometry and a variety of other biochemical and immunological criteria. We demonstrated by NMR analysis that the Pyrl of pRNase 3 forms hydrogen bonds with Lys9 and Ile96 and stabilizes the N-terminal alpha-helix in a rigid conformation. In contrast, the N-terminal alpha-helix becomes flexible and the pK(a) values of the catalytic residues His10 and His97 altered when Pyrl formation is blocked by an extra methionine at the N terminus in the recombinant mqRNase 3. Thus, our results provide a mechanistic explanation on the essential role of Pyrl in maintaining the structural integrity, especially at the N-terminal alpha-helix, and in providing the proper environment for the ionization of His10 and His97 residues for catalysis and cytotoxicity against HeLa. cells. (c) 2005 Elsevier Ltd. All rights reserved.