1c8w: Difference between revisions
New page: left|200px<br /><applet load="1c8w" size="450" color="white" frame="true" align="right" spinBox="true" caption="1c8w, resolution 1.8Å" /> '''THR45GLY VARIANT OF R... |
No edit summary |
||
| Line 1: | Line 1: | ||
[[Image:1c8w.gif|left|200px]]<br /><applet load="1c8w" size=" | [[Image:1c8w.gif|left|200px]]<br /><applet load="1c8w" size="350" color="white" frame="true" align="right" spinBox="true" | ||
caption="1c8w, resolution 1.8Å" /> | caption="1c8w, resolution 1.8Å" /> | ||
'''THR45GLY VARIANT OF RIBONUCLEASE A'''<br /> | '''THR45GLY VARIANT OF RIBONUCLEASE A'''<br /> | ||
==Overview== | ==Overview== | ||
Ribonuclease A (RNase A) catalyzes the cleavage of RNA after pyrimidine | 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== | ==About this Structure== | ||
1C8W is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Bos_taurus Bos taurus] with ACT and CL as [http://en.wikipedia.org/wiki/ligands ligands]. Full crystallographic information is available from [http:// | 1C8W is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Bos_taurus Bos taurus] with <scene name='pdbligand=ACT:'>ACT</scene> and <scene name='pdbligand=CL:'>CL</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1C8W OCA]. | ||
==Reference== | ==Reference== | ||
| Line 13: | Line 13: | ||
[[Category: Bos taurus]] | [[Category: Bos taurus]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
[[Category: Kelemen, B | [[Category: Kelemen, B R.]] | ||
[[Category: Raines, R | [[Category: Raines, R T.]] | ||
[[Category: Schultz, L | [[Category: Schultz, L W.]] | ||
[[Category: Sweeney, R | [[Category: Sweeney, R T.]] | ||
[[Category: ACT]] | [[Category: ACT]] | ||
[[Category: CL]] | [[Category: CL]] | ||
| Line 22: | Line 22: | ||
[[Category: rnase a]] | [[Category: rnase a]] | ||
''Page seeded by [http:// | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 12:03:38 2008'' | ||
Revision as of 13:03, 21 February 2008
|
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 and 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
Page seeded by OCA on Thu Feb 21 12:03:38 2008