4rsk

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{{STRUCTURE_4rsk| PDB=4rsk | SCENE= }}
{{STRUCTURE_4rsk| PDB=4rsk | SCENE= }}
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'''STRUCTURE OF THE K7A/R10A/K66A VARIANT OF RIBONUCLEASE A COMPLEXED WITH 3'-UMP'''
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===STRUCTURE OF THE K7A/R10A/K66A VARIANT OF RIBONUCLEASE A COMPLEXED WITH 3'-UMP===
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==Overview==
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The active-site cleft of bovine pancreatic ribonuclease A (RNase A) is lined with cationic residues that interact with a bound nucleic acid. Those residues interacting with the phosphoryl groups comprise the P0, P1, and P2 subsites, with the scissile P-O5' bond residing in the P1 subsite. Coulombic interactions between the P0 and P2 subsites and phosphoryl groups of the substrate were characterized previously [Fisher, B. M., Ha, J.-H., and Raines, R. T. (1998) Biochemistry 37, 12121-12132]. Here, the interactions between these subsites and the active-site residues His12 and His119 are described in detail. A protein variant in which the cationic residues in these subsites (Lys66 in the P0 subsite and Lys7 and Arg10 in the P2 subsite) were replaced with alanine was crystallized, both free and with bound 3'-uridine monophosphate (3'-UMP). Structures of K7A/R10A/K66A RNase A and the K7A/R10A/K66A RNase A.3'-UMP complex were determined by X-ray diffraction analysis to resolutions of 2.0 and 2.1 A, respectively. There is little observable change between these structures and that of wild-type RNase A, either free or with bound 3'-cytidine monophosphate. K7A/R10A/K66A RNase A was evaluated for its ability to cleave UpA, a dinucleotide substrate that does not span the P0 or the P2 subsites. In comparison to the wild-type enzyme, the value of kcat was decreased by 5-fold and that of kcat/Km was decreased 10-fold, suggesting that these remote subsites interact with the active site. These interactions were characterized by determining the pKa values of His12 and His119 at 0.018 and 0.142 M Na+, both in wild-type RNase A and the K7A/R10A/K66A variant. The side chains of Lys7, Arg10, and Lys66 depress the pKa values of these histidine residues, and this depression is sensitive to the salt concentration. In addition, the P0 and P2 subsites influence the interaction of His12 and His119 with each other, as demonstrated by changes in the cooperativity that gives rise to microscopic pKa values. Finally, the affinity of 3'-UMP for wild-type RNase A and the K7A/R10A/K66A variant at 0.018 and 0.142 M Na+ was determined by isothermal titration calorimetry. 3'-UMP binds to the variant protein with 5-fold weaker affinity at 0.018 M Na+ and 3-fold weaker affinity at 0.142 M Na+ than it binds to wild-type RNase A. Together these data demonstrate that long-range Coulombic interactions are an important feature in catalysis by RNase A.
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(as it appears on PubMed at http://www.pubmed.gov), where 9860854 is the PubMed ID number.
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{{ABSTRACT_PUBMED_9860854}}
==About this Structure==
==About this Structure==
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[[Category: Ribonuclease some]]
[[Category: Ribonuclease some]]
[[Category: Site-directed mutagenesis]]
[[Category: Site-directed mutagenesis]]
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''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sun May 4 22:29:08 2008''
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''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Jul 3 13:49:43 2008''

Revision as of 10:49, 3 July 2008

Image:4rsk.png

Template:STRUCTURE 4rsk

STRUCTURE OF THE K7A/R10A/K66A VARIANT OF RIBONUCLEASE A COMPLEXED WITH 3'-UMP

Template:ABSTRACT PUBMED 9860854

About this Structure

4RSK is a Single protein structure of sequence from Bos taurus. Full crystallographic information is available from OCA.

Reference

Coulombic effects of remote subsites on the active site of ribonuclease A., Fisher BM, Schultz LW, Raines RT, Biochemistry. 1998 Dec 15;37(50):17386-401. PMID:9860854

Page seeded by OCA on Thu Jul 3 13:49:43 2008

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