Neighboring group participation in the transition state of human purine nucleoside phosphorylase

Andrew S. Murkin, Matthew R. Birck, Agnes Rinaldo-Matthis, Wuxian Shi, Erika A. Taylor, Steven C. Almo, Vern L. Schramm

Research output: Contribution to journalArticle

45 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)5038-5049
Number of pages12
JournalBiochemistry
Volume46
Issue number17
DOIs
StatePublished - May 1 2007

Fingerprint

Purine-Nucleoside Phosphorylase
Isotopes
Inosine
Hydroxyl Radical
Kinetics
Hydrogen Bonding
Hydrogen bonds
Crystal structure
X-Rays
X rays
Mutation
Enzymes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Neighboring group participation in the transition state of human purine nucleoside phosphorylase. / Murkin, Andrew S.; Birck, Matthew R.; Rinaldo-Matthis, Agnes; Shi, Wuxian; Taylor, Erika A.; Almo, Steven C.; Schramm, Vern L.

In: Biochemistry, Vol. 46, No. 17, 01.05.2007, p. 5038-5049.

Research output: Contribution to journalArticle

Murkin, Andrew S. ; Birck, Matthew R. ; Rinaldo-Matthis, Agnes ; Shi, Wuxian ; Taylor, Erika A. ; Almo, Steven C. ; Schramm, Vern L. / Neighboring group participation in the transition state of human purine nucleoside phosphorylase. In: Biochemistry. 2007 ; Vol. 46, No. 17. pp. 5038-5049.
@article{2ac0515d8a074c6c81d53738aad5f12e,
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 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.",
author = "Murkin, {Andrew S.} and Birck, {Matthew R.} and Agnes Rinaldo-Matthis and Wuxian Shi and Taylor, {Erika A.} and Almo, {Steven C.} and Schramm, {Vern L.}",
year = "2007",
month = "5",
day = "1",
doi = "10.1021/bi700147b",
language = "English (US)",
volume = "46",
pages = "5038--5049",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "17",

}

TY - JOUR

T1 - Neighboring group participation in the transition state of human purine nucleoside phosphorylase

AU - Murkin, Andrew S.

AU - Birck, Matthew R.

AU - Rinaldo-Matthis, Agnes

AU - Shi, Wuxian

AU - Taylor, Erika A.

AU - Almo, Steven C.

AU - Schramm, Vern L.

PY - 2007/5/1

Y1 - 2007/5/1

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=34247603923&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=34247603923&partnerID=8YFLogxK

U2 - 10.1021/bi700147b

DO - 10.1021/bi700147b

M3 - Article

C2 - 17407325

AN - SCOPUS:34247603923

VL - 46

SP - 5038

EP - 5049

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 17

ER -