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 Pyr1 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 pKa values of the catalytic residues His10 and His97 altered when Pyr1 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 Pyr1 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.
Lou
Yuan-Chao
YC
Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan, ROC.
Huang
Yu-Chie
YC
Pan
Yun-Ru
YR
Chen
Chinpan
C
Liao
You-Di
YD
D000818:Animals; D020134:Catalytic Domain; D002470:Cell Survival / Q000187:drug effects; D047150:Eosinophil Cationic Protein / Q000737:chemistry* / Q000494:pharmacology; D006367:HeLa Cells; D006639:Histidine / Q000737:chemistry; D006801:Humans; D006860:Hydrogen Bonding; D007532:Isoleucine / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011761:Pyrrolidonecarboxylic Acid / Q000737:chemistry*; D011892:Rana catesbeiana; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology
D011994:Recombinant Proteins; D007532:Isoleucine; D006639:Histidine; D047150:Eosinophil Cationic Protein; D008239:Lysine; D011761:Pyrrolidonecarboxylic Acid