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| {{STRUCTURE_2oc9| PDB=2oc9 | SCENE= }} | | {{STRUCTURE_2oc9| PDB=2oc9 | SCENE= }} |
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| '''Crystal stucture of human purine nucleoside phosphorylase mutant H257G with Imm-H'''
| | ===Crystal stucture of human purine nucleoside phosphorylase mutant H257G with Imm-H=== |
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| ==Overview==
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| 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 x DADMe-ImmH x SO4 structures, His257Phe x ImmH x 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'-(3)H 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'-(3)H 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 line below this paragraph, {{ABSTRACT_PUBMED_17407325}}, adds the Publication Abstract to the page |
| | (as it appears on PubMed at http://www.pubmed.gov), where 17407325 is the PubMed ID number. |
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| | {{ABSTRACT_PUBMED_17407325}} |
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| ==About this Structure== | | ==About this Structure== |
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| [[Category: Schramm, V L.]] | | [[Category: Schramm, V L.]] |
| [[Category: Purine nucleoside phosphorylase]] | | [[Category: Purine nucleoside phosphorylase]] |
| ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sun May 4 10:35:34 2008'' | | |
| | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sun Jul 27 21:27:33 2008'' |