1c8w
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THR45GLY VARIANT OF RIBONUCLEASE A
OverviewOverview
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.
About this StructureAbout this Structure
1C8W is a Single protein structure of sequence from Bos taurus with ACT and CL as ligands. Full crystallographic information is available from OCA.
ReferenceReference
Excavating an active site: the nucleobase specificity of ribonuclease A., Kelemen BR, Schultz LW, Sweeney RY, Raines RT, Biochemistry. 2000 Nov 28;39(47):14487-94. PMID:11087402
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