title: Role of histidine-40 in ribonuclease T1 catalysis: three-dimensionalstructures of the partially active His40Lys mutant.
abstract: Histidine-40 is known to participate in phosphodiester transesterification catalyzed by the enzyme ribonuclease T1. A mutant enzyme with a lysine replacing the histidine-40 (His40Lys RNase T1) retains considerable catalytic activity [Steyaert, J., Hallenga, K., Wyns, L., & Stanssens, P. (1990) Biochemistry 29, 9064-9072]. We report on the crystal structures of His40Lys RNase T1 containing a phosphate anion and a guanosine 2'-phosphate inhibitor in the active site, respectively. Similar to previously described structures, the phosphate-containing crystals are of space group P2(1)2(1)2(1), with one molecule per asymmetric unit (a = 48.27 A, b = 46.50 A, c = 41.14 A). The complex with 2'-GMP crystallized in the lower symmetry space group P2(1), with two molecules per asymmetric unit (a = 49.20 A, b = 48.19 A, c = 40.16 A, beta = 90.26). The crystal structures have been solved at 1.8- and 2.0-A resolution yielding R values of 14.5% and 16.0%, respectively. Comparison of these His40Lys structures with the corresponding wild-type structures, containing 2'-GMP [Arni, R., Heinemann, U., Tokuoka, R., & Saenger, W. (1988) J. Biol. Chem. 263, 15358-15368] and vanadate [Kostrewa, D., Hui-Woog Choe, Heinemann, U., & Saenger, W. (1989) Biochemistry 28, 7692-7600] in the active site, respectively, leads to the following conclusions. First, the His40Lys mutation causes no significant changes in the overall structure of RNase T1; second, the Lys40 side chains in the mutant structures occupy roughly the same space as His40 in the corresponding wild-type RNase T1 structures.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Instituut voor Moleculaire Biologie, Vrije Universiteit Brussel, Belgium.
authors.forename: I
authors.initials: I
authors.lastname: Zegers
authors.forename: P
authors.initials: P
authors.lastname: Verhelst
authors.forename: H W
authors.initials: HW
authors.lastname: Choe
authors.forename: J
authors.initials: J
authors.lastname: Steyaert
authors.forename: U
authors.initials: U
authors.lastname: Heinemann
authors.forename: W
authors.initials: W
authors.lastname: Saenger
authors.forename: L
authors.initials: L
authors.lastname: Wyns
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D006639:Histidine / Q000737:chemistry / Q000378:metabolism*; D008670:Metals / Q000378:metabolism; D011487:Protein Conformation; D006163:Ribonuclease T1 / Q000737:chemistry / Q000378:metabolism*; D014961:X-Ray Diffraction
chemical_list: D008670:Metals; D006639:Histidine; D006163:Ribonuclease T1