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1c8w
From Proteopedia
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'''THR45GLY VARIANT OF RIBONUCLEASE A''' | '''THR45GLY VARIANT OF RIBONUCLEASE A''' | ||
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[[Category: Schultz, L W.]] | [[Category: Schultz, L W.]] | ||
[[Category: Sweeney, R T.]] | [[Category: Sweeney, R T.]] | ||
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| - | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Fri May 2 12:28:29 2008'' | |
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Revision as of 09:28, 2 May 2008
THR45GLY VARIANT OF RIBONUCLEASE A
Overview
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 Structure
1C8W is a Single protein structure of sequence from Bos taurus. Full crystallographic information is available from OCA.
Reference
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 Page seeded by OCA on Fri May 2 12:28:29 2008
