2on6
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Crystal stucture of human purine nucleoside phosphorylase mutant H257F 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.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 StructureAbout this Structure
2ON6 is a Single protein structure of sequence from Homo sapiens with as ligand. Active as Purine-nucleoside phosphorylase, with EC number 2.4.2.1 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 Steven C Almo, Schramm VL, Biochemistry. 2007 May 1;46(17):5038-5049. Epub 2007 Apr 4. PMID:17407325
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