Crystal structure of ribonuclease T1 carboxymethylated at Glu58 in complex with 2'-GMP.
The carboxymethylation of RNase T1 at the gamma-carboxyl group of Glu58 leads to a complete loss of the enzymatic activity while it retains substrate-binding ability. Accompanying the carboxymethylation, RNase T1 undergoes a remarkable thermal stabilization of 9 degrees C in the melting temperature (Tm). 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 A resolution. The structure, including 79 water molecules and two Na+, was refined to an R factor of 0.194 with 10 354 reflections > 1 sigma (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.
Ishikawa
K
K
Central Research Laboratories, Ajinomoto Company, Inc., Kawasaki, Japan.
Suzuki
E
E
Tanokura
M
M
Takahashi
K
K
D001665:Binding Sites; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D006157:Guanosine Monophosphate / Q000737:chemistry*; D006860:Hydrogen Bonding; D008958:Models, Molecular; D011487:Protein Conformation; D006163:Ribonuclease T1 / Q000737:chemistry*
C026693:2'-guanylic acid; D006157:Guanosine Monophosphate; D006163:Ribonuclease T1