Structural analysis of an RNase T-1 variant with an altered guanine binding segment

The ribonuclease T-1 variant 9/5 with a guanine recognition segment, altere d from the wild-type amino acid sequence 41-KYNNYE-46 to 41-EFRNWQ-46, has been cocrystallised with the specific inhibitor 2'-GMP. The crystal structu re has been refined to a crystallographic R factor of 0.198 at 2.3 Angstrom resolution. Despite a size reduction of the binding pocket, pushing the in hibitor outside by 1 Angstrom, 2'-GMP is fixed to the primary recognition s ite due to increased aromatic stacking interactions. The phosphate group of 2'-GMP is located about 4.2 Angstrom apart from its position in wild-type ribonuclease T-1-2'-GMP complexes, allowing a Ca2+, coordinating this phosp hate group, to enter the binding pocket. The crystallographic data can be a ligned with the kinetic characterisation of the variant, showing a reductio n of both, guanine affinity and turnover rate. The presence of Ca2+ was sho wn to inhibit variant 9/5 and wild-type enzyme to nearly the same extent. ( C) 1999 Academic Press.