Conformational states of human purine nucleoside phosphorylase at rest, at work, and with transition state analogues

Achelle A. Edwards, Jeremiah D. Tipton, Michael D. Brenowitz, Mark R. Emmett, Alan G. Marshall, Gary B. Evans, Peter C. Tyler, Vern L. Schramm

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

Human purine nucleoside Phosphorylase (PNP) is a homotrimer binding tightly to the transition state analogues Immucillin-H (ImmH; Kd = 56 pM) and DATMe-ImmH-Immucillin-H (DATMe-ImmH; Kd = 8.6 pM). ImmH binds with a larger entropie penalty than DATMe-ImmH, a chemically more flexible inhibitor. The testable hypothesis is that PNP conformational states are more relaxed (dynamic) with DATMe-ImmH, despite tighter binding than with ImmH. PNP conformations are probed by peptide amide deuterium exchange (HDX) using liquid chromatography high-resolution Fourier transform ion cyclotron resonance mass spectrometry and by sedimentation rates. Catalytically equilibrating Michaelis complexes (PNP·PO4·inosine PNP·Hx·R-1-P) and inhibited complexes (PNP·PO4·DATMe-ImmH and PNP·PO4·ImmH) show protection from HDX at 9, 13, and 15 sites per subunit relative to resting PNP (PNP·PO4) in extended incubations. The PNP·PO4· ImmH complex is more compact (by sedimentation rate) than the other complexes. HDX kinetic analysis of ligand-protected sites corresponds to peptides near the catalytic sites. HDX and sedimentation results establish that PNP protein conformation (dynamic motion) correlates more closely with entropy of binding than with affinity. Catalytically active turnover with saturated substrate sites causes less change in HDX and sedimentation rates than binding of transition state analogues. DATMe-ImmH more closely mimics the transition of human PNP than does ImmH and achieves strong binding interactions at the catalytic site while causing relatively modest alterations of the protein dynamic motion. Transition state analogues causing the most rigid, closed protein conformation are therefore not necessarily the most tightly bound. Close mimics of the transition state are hypothesized to retain enzymatic dynamic motions related to transition state formation.

Original languageEnglish (US)
Pages (from-to)2058-2067
Number of pages10
JournalBiochemistry
Volume49
Issue number9
DOIs
StatePublished - Mar 9 2010

Fingerprint

Purine-Nucleoside Phosphorylase
Sedimentation
Conformations
Protein Conformation
Catalytic Domain
Cyclotrons
Cyclotron resonance
Peptides
Proteins
Deuterium
Liquid chromatography
Entropy
Fourier Analysis
Amides
Liquid Chromatography
Mass spectrometry
Mass Spectrometry
Fourier transforms
Ions
Ligands

ASJC Scopus subject areas

  • Biochemistry

Cite this

Conformational states of human purine nucleoside phosphorylase at rest, at work, and with transition state analogues. / Edwards, Achelle A.; Tipton, Jeremiah D.; Brenowitz, Michael D.; Emmett, Mark R.; Marshall, Alan G.; Evans, Gary B.; Tyler, Peter C.; Schramm, Vern L.

In: Biochemistry, Vol. 49, No. 9, 09.03.2010, p. 2058-2067.

Research output: Contribution to journalArticle

Edwards, Achelle A. ; Tipton, Jeremiah D. ; Brenowitz, Michael D. ; Emmett, Mark R. ; Marshall, Alan G. ; Evans, Gary B. ; Tyler, Peter C. ; Schramm, Vern L. / Conformational states of human purine nucleoside phosphorylase at rest, at work, and with transition state analogues. In: Biochemistry. 2010 ; Vol. 49, No. 9. pp. 2058-2067.
@article{d31d315b3cea4e93aaa48764fc063d85,
title = "Conformational states of human purine nucleoside phosphorylase at rest, at work, and with transition state analogues",
abstract = "Human purine nucleoside Phosphorylase (PNP) is a homotrimer binding tightly to the transition state analogues Immucillin-H (ImmH; Kd = 56 pM) and DATMe-ImmH-Immucillin-H (DATMe-ImmH; Kd = 8.6 pM). ImmH binds with a larger entropie penalty than DATMe-ImmH, a chemically more flexible inhibitor. The testable hypothesis is that PNP conformational states are more relaxed (dynamic) with DATMe-ImmH, despite tighter binding than with ImmH. PNP conformations are probed by peptide amide deuterium exchange (HDX) using liquid chromatography high-resolution Fourier transform ion cyclotron resonance mass spectrometry and by sedimentation rates. Catalytically equilibrating Michaelis complexes (PNP·PO4·inosine PNP·Hx·R-1-P) and inhibited complexes (PNP·PO4·DATMe-ImmH and PNP·PO4·ImmH) show protection from HDX at 9, 13, and 15 sites per subunit relative to resting PNP (PNP·PO4) in extended incubations. The PNP·PO4· ImmH complex is more compact (by sedimentation rate) than the other complexes. HDX kinetic analysis of ligand-protected sites corresponds to peptides near the catalytic sites. HDX and sedimentation results establish that PNP protein conformation (dynamic motion) correlates more closely with entropy of binding than with affinity. Catalytically active turnover with saturated substrate sites causes less change in HDX and sedimentation rates than binding of transition state analogues. DATMe-ImmH more closely mimics the transition of human PNP than does ImmH and achieves strong binding interactions at the catalytic site while causing relatively modest alterations of the protein dynamic motion. Transition state analogues causing the most rigid, closed protein conformation are therefore not necessarily the most tightly bound. Close mimics of the transition state are hypothesized to retain enzymatic dynamic motions related to transition state formation.",
author = "Edwards, {Achelle A.} and Tipton, {Jeremiah D.} and Brenowitz, {Michael D.} and Emmett, {Mark R.} and Marshall, {Alan G.} and Evans, {Gary B.} and Tyler, {Peter C.} and Schramm, {Vern L.}",
year = "2010",
month = "3",
day = "9",
doi = "10.1021/bi902041j",
language = "English (US)",
volume = "49",
pages = "2058--2067",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "9",

}

TY - JOUR

T1 - Conformational states of human purine nucleoside phosphorylase at rest, at work, and with transition state analogues

AU - Edwards, Achelle A.

AU - Tipton, Jeremiah D.

AU - Brenowitz, Michael D.

AU - Emmett, Mark R.

AU - Marshall, Alan G.

AU - Evans, Gary B.

AU - Tyler, Peter C.

AU - Schramm, Vern L.

PY - 2010/3/9

Y1 - 2010/3/9

N2 - Human purine nucleoside Phosphorylase (PNP) is a homotrimer binding tightly to the transition state analogues Immucillin-H (ImmH; Kd = 56 pM) and DATMe-ImmH-Immucillin-H (DATMe-ImmH; Kd = 8.6 pM). ImmH binds with a larger entropie penalty than DATMe-ImmH, a chemically more flexible inhibitor. The testable hypothesis is that PNP conformational states are more relaxed (dynamic) with DATMe-ImmH, despite tighter binding than with ImmH. PNP conformations are probed by peptide amide deuterium exchange (HDX) using liquid chromatography high-resolution Fourier transform ion cyclotron resonance mass spectrometry and by sedimentation rates. Catalytically equilibrating Michaelis complexes (PNP·PO4·inosine PNP·Hx·R-1-P) and inhibited complexes (PNP·PO4·DATMe-ImmH and PNP·PO4·ImmH) show protection from HDX at 9, 13, and 15 sites per subunit relative to resting PNP (PNP·PO4) in extended incubations. The PNP·PO4· ImmH complex is more compact (by sedimentation rate) than the other complexes. HDX kinetic analysis of ligand-protected sites corresponds to peptides near the catalytic sites. HDX and sedimentation results establish that PNP protein conformation (dynamic motion) correlates more closely with entropy of binding than with affinity. Catalytically active turnover with saturated substrate sites causes less change in HDX and sedimentation rates than binding of transition state analogues. DATMe-ImmH more closely mimics the transition of human PNP than does ImmH and achieves strong binding interactions at the catalytic site while causing relatively modest alterations of the protein dynamic motion. Transition state analogues causing the most rigid, closed protein conformation are therefore not necessarily the most tightly bound. Close mimics of the transition state are hypothesized to retain enzymatic dynamic motions related to transition state formation.

AB - Human purine nucleoside Phosphorylase (PNP) is a homotrimer binding tightly to the transition state analogues Immucillin-H (ImmH; Kd = 56 pM) and DATMe-ImmH-Immucillin-H (DATMe-ImmH; Kd = 8.6 pM). ImmH binds with a larger entropie penalty than DATMe-ImmH, a chemically more flexible inhibitor. The testable hypothesis is that PNP conformational states are more relaxed (dynamic) with DATMe-ImmH, despite tighter binding than with ImmH. PNP conformations are probed by peptide amide deuterium exchange (HDX) using liquid chromatography high-resolution Fourier transform ion cyclotron resonance mass spectrometry and by sedimentation rates. Catalytically equilibrating Michaelis complexes (PNP·PO4·inosine PNP·Hx·R-1-P) and inhibited complexes (PNP·PO4·DATMe-ImmH and PNP·PO4·ImmH) show protection from HDX at 9, 13, and 15 sites per subunit relative to resting PNP (PNP·PO4) in extended incubations. The PNP·PO4· ImmH complex is more compact (by sedimentation rate) than the other complexes. HDX kinetic analysis of ligand-protected sites corresponds to peptides near the catalytic sites. HDX and sedimentation results establish that PNP protein conformation (dynamic motion) correlates more closely with entropy of binding than with affinity. Catalytically active turnover with saturated substrate sites causes less change in HDX and sedimentation rates than binding of transition state analogues. DATMe-ImmH more closely mimics the transition of human PNP than does ImmH and achieves strong binding interactions at the catalytic site while causing relatively modest alterations of the protein dynamic motion. Transition state analogues causing the most rigid, closed protein conformation are therefore not necessarily the most tightly bound. Close mimics of the transition state are hypothesized to retain enzymatic dynamic motions related to transition state formation.

UR - http://www.scopus.com/inward/record.url?scp=77749337724&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=77749337724&partnerID=8YFLogxK

U2 - 10.1021/bi902041j

DO - 10.1021/bi902041j

M3 - Article

VL - 49

SP - 2058

EP - 2067

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 9

ER -