CRYSTALLOGRAPHIC STUDY OF GLU58ALA RNASE T1-CENTER-DOT-2'-GUANOSINE MONOPHOSPHATE AT 1.9-ANGSTROM RESOLUTION

Glu58 is known to participate in phosphodiester transesterification ca talyzed by the enzyme RNase T1. For Glu58 RNase T1, an altered mechani sm has been proposed in which His40 replaces Glu58 as the base catalys t [Steyaert, J., Hallenga, K., Wyns, L., & Stanssens, P. (1990) Bioche mistry 29, 9064-9072]. Glu58Ala Rnase T1 has been cocrystallized with guanosine 2'-monophosphate (2'-GMP). The crystals are of space group P 2(1), with one molecule per asymmetric unit (a = 32.44 Angstrom, b = 4 9.64 Angstrom, c = 26.09 Angstrom, beta = 99. 17 degrees). The three-d imensional structure of the enzyme was determined to a nominal resolut ion of 1.9 Angstrom, yielding a crystallographic R factor of 0.178 for all X-ray data. Comparison of this structure with wild-type structure s leads to the following conclusions. The minor changes apparent in th e tertiary structure can be explained by either the mutation of Glu58 or by the change in the space group. In the active site, the extra spa ce available through the mutation of Glu58 is occupied by the phosphat e group (after a reorientation) and by a solvent molecule replacing a carboxylate oxygen of Glu58. This solvent molecule is a candidate for participation in the altered mechanism of this mutant enzyme. Followin g up on a study of conserved water sites in RNase T1 crystallized in s pace group P2(1)2(1)2(1) [Malin, R., Zielenkiewicz, P., & Saenger, W. (1991) J. Mol. Biol. 266, 4848-4852], we investigated the hydration st ructure for four different packing modes of RNase T1. Several of the w ater molecules as well as the cation binding site that are conserved i n space group P2(1)2(1)2(1) are absent in structures in other space gr oups.