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Title: Neighboring Group Participation in the Transition State of Human Purine Nucleoside Phosphorylase

Abstract

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,more » 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.« less

Authors:
; ; ; ; ; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
930454
Report Number(s):
BNL-81206-2008-JA
Journal ID: ISSN 0006-2960; TRN: US200904%%725
DOE Contract Number:  
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry; Journal Volume: 46
Country of Publication:
United States
Language:
English
Subject:
08 HYDROGEN; 36 MATERIALS SCIENCE; AFFINITY; CHEMICAL BONDS; CHEMICAL REACTION KINETICS; CRYSTAL STRUCTURE; ENZYMES; HYDROGEN; INOSINE; ISOTOPE EFFECTS; KINETICS; MUTANTS; MUTATIONS; NUCLEOSIDES; PHOSPHOTRANSFERASES; POSITIONING; PURINES; national synchrotron light source

Citation Formats

Murkin,A., Birck, M., Rinaldo-Matthis, A., Shi, W., Taylor, E., Almo, S., and Schramm, V. Neighboring Group Participation in the Transition State of Human Purine Nucleoside Phosphorylase. United States: N. p., 2007. Web. doi:10.1021/bi700147b.
Murkin,A., Birck, M., Rinaldo-Matthis, A., Shi, W., Taylor, E., Almo, S., & Schramm, V. Neighboring Group Participation in the Transition State of Human Purine Nucleoside Phosphorylase. United States. doi:10.1021/bi700147b.
Murkin,A., Birck, M., Rinaldo-Matthis, A., Shi, W., Taylor, E., Almo, S., and Schramm, V. Mon . "Neighboring Group Participation in the Transition State of Human Purine Nucleoside Phosphorylase". United States. doi:10.1021/bi700147b.
@article{osti_930454,
title = {Neighboring Group Participation in the Transition State of Human Purine Nucleoside Phosphorylase},
author = {Murkin,A. and Birck, M. and Rinaldo-Matthis, A. and Shi, W. and Taylor, E. and Almo, S. and Schramm, V.},
abstractNote = {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.},
doi = {10.1021/bi700147b},
journal = {Biochemistry},
number = ,
volume = 46,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}