Crystal Structure of Ribonuclease T1 Carboxymethylated at Glu58 in Complex with 2′-GMP ‡ Kohki Ishikawa § and Ei-ichiro Suzuki* Central Research Laboratories, Ajinomoto Company, Inc., Suzuki-cho, Kawasaki-ku, Kawasaki 210, Japan Masaru Tanokura Biotechnology Research Center, UniVersity of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113, Japan Kenji Takahashi Tokyo UniVersity of Pharmacy and Life Science, Horinouchi, Hachioji, Tokyo 192-03, Japan ReceiVed February 28, 1996; ReVised Manuscript ReceiVed April 26, 1996 X ABSTRACT: The carboxymethylation of RNase T1 at the γ-carboxyl group of Glu58 leads to a complete loss of the enzymatic activity while it retains substrate-binding ability. Accompanying the carboxy- methylation, RNase T1 undergoes a remarkable thermal stabilization of 9 °C in the melting temperature (T m ). In order to clarify the inactivation and stabilization mechanisms of RNase T1 by carboxymethylation, the crystal structure of carboxymethylated RNase T1 (CM-RNase T1) complexed with 2′-GMP was determined at 1.8 Å resolution. The structure, including 79 water molecules and two Na + , was refined to an R factor of 0.194 with 10 354 reflections >1σ(F). The carboxyl group of CM-Glu58, which locates in the active site, occupies almost the same position as the phosphate group of 2′-GMP in the crystal structure of intact RNase T1‚2′-GMP complex. Therefore, the phosphate group of 2′-GMP cannot locate in the active site but protrudes toward the solvent. This forces 2′-GMP to adopt an anti form, which contrasts with the syn form in the crystal of the intact RNase T1‚2′-GMP complex. The inaccessibility of the phosphate group to the active site can account for the lack of the enzymatic activity in CM-RNase T1. One of the carboxyl oxygen atoms of CM-Glu58 forms two hydrogen bonds with the side-chains of Tyr38 and His40. These hydrogen bonds are considered to mainly contribute to the higher thermal stability of CM-RNase T1. Another carboxyl oxygen atoms of CM-Glu58 is situated nearby His40 and Arg77. This may provide additional electrostatic stabilization. Ribonuclease T1 (RNase T1; 1 EC 3.1.27.3) from the fungus Aspergillus oryzae is an enzyme that specifically hydrolyzes a phosphate diester bond at the 3′-side of guanosine in single-stranded RNA (Sato & Egami, 1957). RNase T1 is a globular protein with 104 amino acid residues (M r 11 085) in a single polypeptide chain (Takahashi, 1965). Since RNase T1 is a small, stable enzyme, a number of studies, including chemical modifications (Takahashi & Moore, 1982), protein engineering (Nishikawa et al., 1987; Steyaert et al., 1990, etc.), NMR (Inagaki et al., 1981, etc.), and X-ray crystallography (Heinemann & Saenger, 1982, etc.), have been performed on this protein. These studies have elucidated that His40, Glu58, Arg77, and His92 are involved in the active site. As regards the crystallographic studies, many crystal structures, the free form (Martinez- Oyanedel et al., 1991), and the complexed forms with 2′- guanylic acid (2′-GMP; Arni et al., 1987; Sugio et al., 1988), 3′-guanylic acid (3′-GMP; Heydenreich et al., 1993; Gohda et al., 1994), guanylyl-2′,5′-guanosine (2′,5′-GpG; Koepke et al., 1989), guanosine-3′,5′-bisphosphate (3′,5′-pGp; Lenz et al., 1993), and vanadate (H 2 VO 4 - ; Kostrewa et al., 1989), have been determined. These crystal structures have played especially important roles in elucidating the enzymatic mechanism and the nucleotide binding modes. Furthermore, some mutant proteins of RNase T1 have been subjected to crystallographic studies to allow more detailed discussions on this enzyme [for example, Tyr45Trp complexed with 2′- AMP and 2′-GMP (Hakoshima et al., 1992)]. Takahashi et al. (1967) reported that RNase T1 is completely inactivated by the selective carboxymethyla- tion of the γ-carboxyl group of Glu58 (-COO - f -COOCH2COO - ) with iodoacetate at pH 5.5. The Glu58 carboxylate has been proposed to act as base in a general acid-base catalysis (Takahashi, 1970a; Heydenreich et al., 1993). Despite the loss of the enzymatic activity, carboxy- methylated RNase T1 (CM-RNase T1) retains almost the same binding ability against guanosine and 10% binding ability against 2′-GMP as compared to intact RNase T1, in terms of the K d values (Takahashi & Moore, 1982). This suggested that the carboxymethylation of Glu58 affects only the binding of phosphate portion and hardly affects that of guanosine. To compare the thermal stability of CM-RNase T1 with that of intact RNase T1, 1 H NMR spectra measurement was performed at various temperatures and the changes in the peak areas were plotted for two specific protons (Kojima et al., 1994). It turned out that the T m value of CM-RNase T1 is higher than that of intact RNase T1 by 9 °C(∆∆G ) 5.25 kcal/mol). Kojima et al. (1994) ascribed this remarkable ‡ The atomic coordinates and structure factors have been deposited with the Protein Data Bank (ID Code 1DET). * To whom correspondence should be addressed. § University of Tokyo. X Abstract published in AdVance ACS Abstracts, June 1, 1996. 1 Abbreviations: CM-RNase T1; ribonuclease T1 carboxymethylated at the γ-carboxyl group of Glu58; 2′-GMP; 2′-guanylic acid; Tm, melting temperature. 8329 Biochemistry 1996, 35, 8329-8334 S0006-2960(96)00493-X CCC: $12.00 © 1996 American Chemical Society