2oc4
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| , resolution 2.592Å | |||||||
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| Ligands: | , | ||||||
| Gene: | NP, PNP (Homo sapiens) | ||||||
| Activity: | Purine-nucleoside phosphorylase, with EC number 2.4.2.1 | ||||||
| Related: | 2OC9, 2ON6, 2A0X, 2A0Y, 2A0W
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| Resources: | FirstGlance, OCA, PDBsum, RCSB | ||||||
| Coordinates: | save as pdb, mmCIF, xml | ||||||
Crystal stucture of human purine nucleoside phosphorylase mutant H257D with Imm-H
OverviewOverview
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.
About this StructureAbout this Structure
2OC4 is a Single protein structure of sequence from Homo sapiens. Full crystallographic information is available from OCA.
ReferenceReference
Neighboring group participation in the transition state of human purine nucleoside phosphorylase., Murkin AS, Birck MR, Rinaldo-Matthis A, Shi W, Taylor EA, Almo SC, Schramm VL, Biochemistry. 2007 May 1;46(17):5038-49. Epub 2007 Apr 4. PMID:17407325
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