Physicochemical Characterization of the Catalytic Unit of Hammerhead Ribozyme and Its Relationship with the Catalytic Activity
Yoshiyuki Tanaka,
Daichi Yamanaka,
Saori Morioka,
Taishi Yamaguchi,
Masayuki Morikawa,
Takashi S. Kodama,
Vladimír Sychrovský,
Chojiro Kojima,
Yoshikazu Hattori
Affiliations
Yoshiyuki Tanaka
Laboratory of Analytical Chemistry, Department of Pharmaceutical Sciences, Tokushima Bunri University, 180 Nishihama-Boji, Yamashiro-cho, Tokushima 770-8514, Japan
Daichi Yamanaka
Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan
Saori Morioka
Laboratory of Analytical Chemistry, Department of Pharmaceutical Sciences, Tokushima Bunri University, 180 Nishihama-Boji, Yamashiro-cho, Tokushima 770-8514, Japan
Taishi Yamaguchi
Laboratory of Analytical Chemistry, Department of Pharmaceutical Sciences, Tokushima Bunri University, 180 Nishihama-Boji, Yamashiro-cho, Tokushima 770-8514, Japan
Masayuki Morikawa
Laboratory of Analytical Chemistry, Department of Pharmaceutical Sciences, Tokushima Bunri University, 180 Nishihama-Boji, Yamashiro-cho, Tokushima 770-8514, Japan
Takashi S. Kodama
Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita 565-0871, Japan
Vladimír Sychrovský
Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo náměstí 542/2, 166 10 Praha, Czech Republic
Chojiro Kojima
Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita 565-0871, Japan
Yoshikazu Hattori
Laboratory of Analytical Chemistry, Department of Pharmaceutical Sciences, Tokushima Bunri University, 180 Nishihama-Boji, Yamashiro-cho, Tokushima 770-8514, Japan
The catalytic mechanism of hammerhead ribozymes (HHRzs) attracted great attention in relation to the chemical origin of life. However, the basicity (pKa) of the catalytic sites of HHRzs has not been studied so far. As a result, the investigation of the currently assumed mechanism from an experimentally derived pKa value has been impossible. In HHRzs, there exists a highly functionalized structural unit (A9-G10.1 site) with a catalytic residue (G12) for the nucleophile activation and metal ion-binding residue (G10.1). As inferred from this fact, there might be a possibility that HHRzs may utilize specific functions of the A9-G10.1 motif for the catalytic reaction. Therefore, here we studied the basicity of G12/G10.1-corresponding residues using RNA duplexes including the A9-G10.1 motif without other conserved residues of HHRzs. From the pH-titration experiments with NMR spectra, we have obtained the intrinsic basicity of the G12/G10.1-corresponding residues in the motif, with pKa > 11.5 (N1 of G12) and pKa 4.5 (N7 of G10.1) for the first time. Based on the derived irregular basicity, their correlation with a catalytic activity and a metal affinity were investigated. In total, the derived thermodynamic properties are an intrinsic nature of the exclusive catalytic unit of HHRzs, which will be an outstanding pivot point for the mechanistic analyses.