Hakoshima T, Oka K, Toda S, Tanaka M, Goda K, Higo T, Itoh T, Minami H, Tomita K, Nishikawa S
Faculty of Pharmaceutical Sciences, Osaka University, Suita.
J Biochem. 1990 Nov;108(5):695-8. doi: 10.1093/oxfordjournals.jbchem.a123265.
Ribonuclease T1 and the mutant enzymes were cocrystallized with several ribonucleotides, including non-hydrolyzable substrate analogs of di- and triribonucleotides, which have a novel guanylate in which the 2'-hydroxyl group of the ribose is replaced by a fluorine atom. One of the mutant enzymes has a tryptophan residue, instead of Tyr45 of the wild-type enzyme, to enhance the binding of ribonucleotides to the enzyme and the other mutant enzyme has histidine and aspartate residues, instead of Asn43 and Asn44, respectively, to reproduce the natural substitutions found in ribonuclease Ms. Polymorphism of the crystals was observed for wild-type and mutant enzymes. However, orthorhombic crystals, which are virtually all isomorphous to each other, were successfully obtained from wild-type and mutant (Y45W) enzymes by the macroscopic seeding technique using mother crystals of the wild-type ribonuclease T1 complexed with 2'GMP or 3'GMP. The diffraction patterns of these crystals extend beyond 2.5 A resolution and the diffraction data were collected from some of the crystals on a diffractometer up to a range of 2.5 to 1.8 A resolution.
核糖核酸酶T1及突变酶与几种核糖核苷酸共结晶，其中包括二核糖核苷酸和三核糖核苷酸的不可水解底物类似物，这些类似物含有一种新型鸟苷酸，其核糖的2'-羟基被氟原子取代。一种突变酶含有一个色氨酸残基，取代了野生型酶的Tyr45，以增强核糖核苷酸与酶的结合；另一种突变酶分别含有组氨酸和天冬氨酸残基，取代了Asn43和Asn44，以重现核糖核酸酶Ms中发现的天然取代。观察到野生型和突变酶晶体的多态性。然而，通过使用与2'GMP或3'GMP复合的野生型核糖核酸酶T1的母晶体进行宏观接种技术，从野生型和突变（Y45W）酶中成功获得了几乎彼此同晶的正交晶体。这些晶体的衍射图谱延伸至2.5 Å分辨率以上，并且在衍射仪上从一些晶体收集了分辨率范围为2.5至1.8 Å的衍射数据。
