Neighboring Group Participation in the Transition State of Human Purine Nucleoside Phosphorylase
The X-ray crystal structures of human purine nucleoside phosphorylase (PNP) with bound inosine or transition-state analogues show His{sup 257} 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 (K{sub m}/K{sub i}) 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 His{sup 257} serves an important role in the early stages of transition-state formation. Whereas mutation of His{sup 257} resulted in little variation in the PNP{center_dot}DADMe-ImmH{center_dot}SO{sub 4} structures, His257Phe{center_dot}ImmH{center_dot}PO{sub 4} 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'-{sup 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'-{sup 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.
- Research Organization:
- Brookhaven National Lab. (BNL), Upton, NY (United States). National Synchrotron Light Source
- Sponsoring Organization:
- Doe - Office Of Science
- DOE Contract Number:
- DE-AC02-98CH10886
- OSTI ID:
- 930454
- Report Number(s):
- BNL-81206-2008-JA; TRN: US200904%%725
- Journal Information:
- Biochemistry, Vol. 46; ISSN 0006-2960
- Country of Publication:
- United States
- Language:
- English
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