2oc9: Difference between revisions
New page: left|200px<br /> <applet load="2oc9" size="450" color="white" frame="true" align="right" spinBox="true" caption="2oc9, resolution 2.590Å" /> '''Crystal stucture o... |
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[[Image:2oc9. | [[Image:2oc9.jpg|left|200px]]<br /><applet load="2oc9" size="350" color="white" frame="true" align="right" spinBox="true" | ||
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caption="2oc9, resolution 2.590Å" /> | caption="2oc9, resolution 2.590Å" /> | ||
'''Crystal stucture of human purine nucleoside phosphorylase mutant H257G with Imm-H'''<br /> | '''Crystal stucture of human purine nucleoside phosphorylase mutant H257G with Imm-H'''<br /> | ||
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==Overview== | ==Overview== | ||
The X-ray crystal structures of human purine nucleoside phosphorylase, (PNP) with bound inosine or transition-state analogues show His257 within, hydrogen bonding distance of the 5'-hydroxyl. The mutants His257Phe, His257Gly, and His257Asp exhibited greatly decreased affinity for, Immucillin-H (ImmH), binding this mimic of an early transition state as, much as 370-fold (Km/Ki) less tightly than native PNP. In contrast, these, mutants bound DADMe-ImmH, a mimic of a late transition state, nearly as, well as the native enzyme. These results indicate that His257 serves an, important role in the early stages of transition-state formation. Whereas, mutation of His257 resulted in little variation in the PNP.DADMe-ImmH.SO4, structures, His257Phe.ImmH.PO4 showed distortion at the 5'-hydroxyl, indicating the importance of H-bonding in positioning this group during, progression to the transition state. Binding isotope effect (BIE) and, kinetic isotope effect (KIE) studies of the remote 5'-3H for the, arsenolysis of inosine with native PNP revealed a BIE of 1.5% and an, unexpectedly large intrinsic KIE of 4.6%. This result is interpreted as a, moderate electronic distortion toward the transition state in the, Michaelis complex with continued development of a similar distortion at, the transition state. The mutants His257Phe, His257Gly, and His257Asp, altered the 5'-3H intrinsic KIE to -3, -14, and 7%, respectively, while, the BIEs contributed 2, 2, and -2%, respectively. These surprising results, establish that forces in the Michaelis complex, reported by the BIEs, can, be reversed or enhanced at the transition state. | The X-ray crystal structures of human purine nucleoside phosphorylase, (PNP) with bound inosine or transition-state analogues show His257 within, hydrogen bonding distance of the 5'-hydroxyl. The mutants His257Phe, His257Gly, and His257Asp exhibited greatly decreased affinity for, Immucillin-H (ImmH), binding this mimic of an early transition state as, much as 370-fold (Km/Ki) less tightly than native PNP. In contrast, these, mutants bound DADMe-ImmH, a mimic of a late transition state, nearly as, well as the native enzyme. These results indicate that His257 serves an, important role in the early stages of transition-state formation. Whereas, mutation of His257 resulted in little variation in the PNP.DADMe-ImmH.SO4, structures, His257Phe.ImmH.PO4 showed distortion at the 5'-hydroxyl, indicating the importance of H-bonding in positioning this group during, progression to the transition state. Binding isotope effect (BIE) and, kinetic isotope effect (KIE) studies of the remote 5'-3H for the, arsenolysis of inosine with native PNP revealed a BIE of 1.5% and an, unexpectedly large intrinsic KIE of 4.6%. This result is interpreted as a, moderate electronic distortion toward the transition state in the, Michaelis complex with continued development of a similar distortion at, the transition state. The mutants His257Phe, His257Gly, and His257Asp, altered the 5'-3H intrinsic KIE to -3, -14, and 7%, respectively, while, the BIEs contributed 2, 2, and -2%, respectively. These surprising results, establish that forces in the Michaelis complex, reported by the BIEs, can, be reversed or enhanced at the transition state. | ||
==About this Structure== | ==About this Structure== | ||
2OC9 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens] with PO4 and IMH as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Purine-nucleoside_phosphorylase Purine-nucleoside phosphorylase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.4.2.1 2.4.2.1] Full crystallographic information is available from [http:// | 2OC9 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens] with <scene name='pdbligand=PO4:'>PO4</scene> and <scene name='pdbligand=IMH:'>IMH</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Purine-nucleoside_phosphorylase Purine-nucleoside phosphorylase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.4.2.1 2.4.2.1] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2OC9 OCA]. | ||
==Reference== | ==Reference== | ||
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[[Category: purine nucleoside phosphorylase]] | [[Category: purine nucleoside phosphorylase]] | ||
''Page seeded by [http:// | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Wed Jan 23 15:12:19 2008'' | ||
