1C8W

THR45GLY VARIANT OF RIBONUCLEASE A

Summary for 1C8W

• Entry DOI: 10.2210/pdb1c8w/pdb
• Descriptor: PROTEIN (Ribonuclease A), ACETATE ION, CHLORIDE ION, ... (4 entities in total)
• Functional Keywords: anti-parallel beta sheet, rnase a, hydrolase
• Biological source: Bos taurus (cattle)
• Cellular location: Secreted: P61823
• Total number of polymer chains: 1
• Total formula weight: 13829.68

Authors

Kelemen, B.R.,Sweeney, R.T.,Schultz, L.W.,Raines, R.T. (deposition date: 1999-07-30, release date: 2002-05-01, Last modification date: 2024-10-30)

Primary citation

Kelemen, B.R.,Schultz, L.W.,Sweeney, R.Y.,Raines, R.T.
Excavating an active site: the nucleobase specificity of ribonuclease A.
Biochemistry, 39:14487-14494, 2000

PubMed 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.

PubMed: 11087402
DOI: 10.1021/bi001862f

Experimental method

X-RAY DIFFRACTION (1.8 Å)