title: Excavating an active site: the nucleobase specificity of ribonuclease A.
abstract: Ribonuclease A (RNase A) catalyzes the cleavage of RNA after pyrimidine nucleotides. When bound in the active site, the base of a pyrimidine nucleotide forms hydrogen bonds with the side chain of Thr45. Here, the role of Thr45 was probed by using the wild-type enzyme, its T45G variant, X-ray diffraction analysis, and synthetic oligonucleotides as ligands and substrates. Catalytic specificity was determined with the fluorogenic substrate: 6-carboxyfluorescein approximately dArXdAdA approximately 6-carboxytetramethylrhodamine (6-FAM approximately dArXdAdA approximately 6-TAMRA), where X = C, U, A, or G. Wild-type RNase A cleaves 10(6)-fold faster when X = C than when X = A. Likewise, its affinity for the non-hydrolyzable oligonucleotide 6-FAM approximately d(CAA) is 50-fold greater than for 6-FAM approximately d(AAA). T45G RNase A cleaves 6-FAM approximately dArAdAdA approximately 6-TAMRA 10(2)-fold faster than does the wild-type enzyme. The structure of crystalline T45G RNase A, determined at 1.8-A resolution by X-ray diffraction analysis, does not reveal new potential interactions with a nucleobase. Indeed, the two enzymes have a similar affinity for 6-FAM approximately d(AAA). The importance of pentofuranosyl ring conformation to nucleotide specificity was probed with 6-FAM approximately d(AU(F)AA), where U(F) is 2'-deoxy-2'-fluorouridine. The conformation of the pentofuranosyl ring in dU(F) is known to be more similar to that in rU than dU. The affinity of wild-type RNase A for 6-FAM approximately d(AU(F)AA) is 50-fold lower than for 6-FAM approximately d(AUAA). This discrimination is lost in the T45G enzyme. Together, these data indicate that the side chain of Thr45 plays multiple roles-interacting favorably with pyrimidine nucleobases but unfavorably with purine nucleobases. Moreover, a ribose-like ring disfavors the interaction of Thr45 with a pyrimidine nucleobase, suggesting that Thr45 enhances catalysis by ground-state destabilization.
authors.affiliation: Department of Biochemistry and Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
authors.forename: B R
authors.initials: BR
authors.lastname: Kelemen
authors.forename: L W
authors.initials: LW
authors.lastname: Schultz
authors.forename: R Y
authors.initials: RY
authors.lastname: Sweeney
authors.forename: R T
authors.initials: RT
authors.lastname: Raines
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002236:Carbohydrate Conformation; D002384:Catalysis; D002417:Cattle; D003460:Crystallization; D018360:Crystallography, X-Ray; D003597:Cytosine Nucleotides / Q000737:chemistry / Q000235:genetics; D005998:Glycine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D011685:Purine Nucleotides / Q000737:chemistry / Q000235:genetics; D011742:Pyrimidine Nucleotides / Q000737:chemistry* / Q000235:genetics; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000235:genetics; D012263:Ribonucleosides / Q000737:chemistry / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics; D013912:Threonine / Q000235:genetics; D014500:Uracil Nucleotides / Q000737:chemistry / Q000235:genetics
chemical_list: D003597:Cytosine Nucleotides; D011685:Purine Nucleotides; D011742:Pyrimidine Nucleotides; D012263:Ribonucleosides; D014500:Uracil Nucleotides; D013912:Threonine; D012259:Ribonuclease, Pancreatic; D005998:Glycine