Ishikawa K, Suzuki E, Tanokura M, Takahashi K
Central Research Laboratories, Ajinomoto Company, Inc., Kawasaki, Japan.
Biochemistry. 1996 Jun 25;35(25):8329-34. doi: 10.1021/bi960493d.
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.
核糖核酸酶T1（RNase T1）在谷氨酸58（Glu58）的γ-羧基上进行羧甲基化后，酶活性完全丧失，但仍保留底物结合能力。伴随着羧甲基化，RNase T1在解链温度（Tm）下有显著的热稳定性提高，达9摄氏度。为阐明羧甲基化对RNase T1的失活和稳定机制，测定了与2'-鸟苷一磷酸（2'-GMP）复合的羧甲基化RNase T1（CM-RNase T1）的晶体结构，分辨率为1.8埃。该结构包括79个水分子和两个钠离子，用10354个大于1σ（F）的反射将R因子精修至0.194。位于活性位点的CM-Glu58的羧基，在完整RNase T1-2'-GMP复合物的晶体结构中占据与2'-GMP的磷酸基团几乎相同的位置。因此，2'-GMP的磷酸基团无法定位在活性位点，而是向溶剂突出。这迫使2'-GMP采取反式构象，这与完整RNase T1-2'-GMP复合物晶体中的顺式构象形成对比。磷酸基团无法进入活性位点可解释CM-RNase T1中缺乏酶活性的原因。CM-Glu58的一个羧基氧原子与酪氨酸38（Tyr38）和组氨酸40（His40）的侧链形成两个氢键。这些氢键被认为主要有助于CM-RNase T1具有更高的热稳定性。CM-Glu58的另一个羧基氧原子位于His40和精氨酸77（Arg77）附近。这可能提供额外的静电稳定作用。
