Ribocation Transition State Capture and Rebound in Human Purine Nucleoside Phosphorylase

Mahmoud Ghanem, Andrew S. Murkin, Vern L. Schramm

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of 6-oxy-purine nucleosides to the corresponding purine base and α-D-ribose 1-phosphate. Its genetic loss causes a lethal T cell deficiency. The highly reactive ribocation transition state of human PNP is protected from solvent by hydrophobic residues that sequester the catalytic site. The catalytic site was enlarged by replacing individual catalytic site amino acids with glycine. Reactivity of the ribocation transition state was tested for capture by water and other nucleophiles. In the absence of phosphate, inosine is hydrolyzed by native, Y88G, F159G, H257G, and F200G enzymes. Phosphorolysis but not hydrolysis is detected when phosphate is bound. An unprecedented N9-to-N3 isomerization of inosine is catalyzed by H257G and F200G in the presence of phosphate and by all PNPs in the absence of phosphate. These results establish a ribocation lifetime too short to permit capture by water. An enlarged catalytic site permits ribocation formation with relaxed geometric constraints, permitting nucleophilic rebound and N3-inosine isomerization.

Original languageEnglish (US)
Pages (from-to)971-979
Number of pages9
JournalChemistry and Biology
Volume16
Issue number9
DOIs
StatePublished - Sep 25 2009

Fingerprint

Purine-Nucleoside Phosphorylase
Catalytic Domain
Inosine
Phosphates
Isomerization
Inosine Nucleotides
Purine Nucleosides
Nucleophiles
Ribose
Water
Glycine
Hydrolysis
Amino Acids
Enzymes

Keywords

  • CHEMBIO
  • PROTEINS

ASJC Scopus subject areas

  • Biochemistry
  • Drug Discovery
  • Molecular Biology
  • Clinical Biochemistry
  • Molecular Medicine
  • Pharmacology

Cite this

Ribocation Transition State Capture and Rebound in Human Purine Nucleoside Phosphorylase. / Ghanem, Mahmoud; Murkin, Andrew S.; Schramm, Vern L.

In: Chemistry and Biology, Vol. 16, No. 9, 25.09.2009, p. 971-979.

Research output: Contribution to journalArticle

@article{a5fdaea090414a06b0e442c1cac1331c,
title = "Ribocation Transition State Capture and Rebound in Human Purine Nucleoside Phosphorylase",
abstract = "Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of 6-oxy-purine nucleosides to the corresponding purine base and α-D-ribose 1-phosphate. Its genetic loss causes a lethal T cell deficiency. The highly reactive ribocation transition state of human PNP is protected from solvent by hydrophobic residues that sequester the catalytic site. The catalytic site was enlarged by replacing individual catalytic site amino acids with glycine. Reactivity of the ribocation transition state was tested for capture by water and other nucleophiles. In the absence of phosphate, inosine is hydrolyzed by native, Y88G, F159G, H257G, and F200G enzymes. Phosphorolysis but not hydrolysis is detected when phosphate is bound. An unprecedented N9-to-N3 isomerization of inosine is catalyzed by H257G and F200G in the presence of phosphate and by all PNPs in the absence of phosphate. These results establish a ribocation lifetime too short to permit capture by water. An enlarged catalytic site permits ribocation formation with relaxed geometric constraints, permitting nucleophilic rebound and N3-inosine isomerization.",
keywords = "CHEMBIO, PROTEINS",
author = "Mahmoud Ghanem and Murkin, {Andrew S.} and Schramm, {Vern L.}",
year = "2009",
month = "9",
day = "25",
doi = "10.1016/j.chembiol.2009.07.012",
language = "English (US)",
volume = "16",
pages = "971--979",
journal = "Cell Chemical Biology",
issn = "2451-9448",
publisher = "Elsevier Inc.",
number = "9",

}

TY - JOUR

T1 - Ribocation Transition State Capture and Rebound in Human Purine Nucleoside Phosphorylase

AU - Ghanem, Mahmoud

AU - Murkin, Andrew S.

AU - Schramm, Vern L.

PY - 2009/9/25

Y1 - 2009/9/25

N2 - Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of 6-oxy-purine nucleosides to the corresponding purine base and α-D-ribose 1-phosphate. Its genetic loss causes a lethal T cell deficiency. The highly reactive ribocation transition state of human PNP is protected from solvent by hydrophobic residues that sequester the catalytic site. The catalytic site was enlarged by replacing individual catalytic site amino acids with glycine. Reactivity of the ribocation transition state was tested for capture by water and other nucleophiles. In the absence of phosphate, inosine is hydrolyzed by native, Y88G, F159G, H257G, and F200G enzymes. Phosphorolysis but not hydrolysis is detected when phosphate is bound. An unprecedented N9-to-N3 isomerization of inosine is catalyzed by H257G and F200G in the presence of phosphate and by all PNPs in the absence of phosphate. These results establish a ribocation lifetime too short to permit capture by water. An enlarged catalytic site permits ribocation formation with relaxed geometric constraints, permitting nucleophilic rebound and N3-inosine isomerization.

AB - Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of 6-oxy-purine nucleosides to the corresponding purine base and α-D-ribose 1-phosphate. Its genetic loss causes a lethal T cell deficiency. The highly reactive ribocation transition state of human PNP is protected from solvent by hydrophobic residues that sequester the catalytic site. The catalytic site was enlarged by replacing individual catalytic site amino acids with glycine. Reactivity of the ribocation transition state was tested for capture by water and other nucleophiles. In the absence of phosphate, inosine is hydrolyzed by native, Y88G, F159G, H257G, and F200G enzymes. Phosphorolysis but not hydrolysis is detected when phosphate is bound. An unprecedented N9-to-N3 isomerization of inosine is catalyzed by H257G and F200G in the presence of phosphate and by all PNPs in the absence of phosphate. These results establish a ribocation lifetime too short to permit capture by water. An enlarged catalytic site permits ribocation formation with relaxed geometric constraints, permitting nucleophilic rebound and N3-inosine isomerization.

KW - CHEMBIO

KW - PROTEINS

UR - http://www.scopus.com/inward/record.url?scp=70349190311&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=70349190311&partnerID=8YFLogxK

U2 - 10.1016/j.chembiol.2009.07.012

DO - 10.1016/j.chembiol.2009.07.012

M3 - Article

C2 - 19778725

AN - SCOPUS:70349190311

VL - 16

SP - 971

EP - 979

JO - Cell Chemical Biology

JF - Cell Chemical Biology

SN - 2451-9448

IS - 9

ER -