Crystal structure of acivicin-inhibited γ-glutamyltranspeptidase reveals critical roles for Its C-Terminus in autoprocessing and catalysis

Kristin Williams, Sierra Cullati, Aaron Sand, Ekaterina I. Biterova, Joseph J. Barycki

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

Helicobacter pylori γ-glutamyltranspeptidase (HpGT) is a general γ-glutamyl hydrolase and a demonstrated virulence factor. The enzyme confers a growth advantage to the bacterium, providing essential amino acid precursors by initiating the degradation of extracellular glutathione and glutamine. HpGT is a member of the N-terminal nucleophile (Ntn) hydrolase superfamily and undergoes autoprocessing to generate the active form of the enzyme. Acivicin is a widely used γ-glutamyltranspeptidase inhibitor that covalently modifies the enzyme, but its precise mechanism of action remains unclear. The time-dependent inactivation of HpGT exhibits a hyperbolic dependence on acivicin concentration with k max = 0.033 ± 0.006 s- 1 and K I = 19.7 ± 7.2 μM. Structure determination of acivicin-modified HpGT (1.7 Å; R factor = 17.9%; R free = 20.8%) demonstrates that acivicin is accommodated within the γ-glutamyl binding pocket of the enzyme. The hydroxyl group of Thr 380, the catalytic nucleophile in the autoprocessing and enzymatic reactions, displaces chloride from the acivicin ring to form the covalently linked complex. Within the acivicin-modified HpGT structure, the C-terminus of the protein becomes ordered with Phe 567 positioned over the active site. Substitution or deletion of Phe 567 leads to a > 10-fold reduction in enzymatic activity, underscoring its importance in catalysis. The mobile C-terminus is positioned by several electrostatic interactions within the C-terminal region, most notably a salt bridge between Arg 475 and Glu 566. Mutational analysis reveals that Arg 475 is critical for the proper placement of the C-terminal region, the Tyr 433 containing loop, and the proposed oxyanion hole.

Original languageEnglish (US)
Pages (from-to)2459-2467
Number of pages9
JournalBiochemistry
Volume48
Issue number11
DOIs
StatePublished - Mar 24 2009

Fingerprint

acivicin
Catalysis
Helicobacter pylori
Crystal structure
Enzymes
Nucleophiles
Essential Amino Acids
R Factors
Hydrolases
Virulence Factors
Protein C
Coulomb interactions
Glutamine
Static Electricity
Hydroxyl Radical
Glutathione
Chlorides
Catalytic Domain
Bacteria
Substitution reactions

ASJC Scopus subject areas

  • Biochemistry

Cite this

Crystal structure of acivicin-inhibited γ-glutamyltranspeptidase reveals critical roles for Its C-Terminus in autoprocessing and catalysis. / Williams, Kristin; Cullati, Sierra; Sand, Aaron; Biterova, Ekaterina I.; Barycki, Joseph J.

In: Biochemistry, Vol. 48, No. 11, 24.03.2009, p. 2459-2467.

Research output: Contribution to journalArticle

Williams, Kristin ; Cullati, Sierra ; Sand, Aaron ; Biterova, Ekaterina I. ; Barycki, Joseph J. / Crystal structure of acivicin-inhibited γ-glutamyltranspeptidase reveals critical roles for Its C-Terminus in autoprocessing and catalysis. In: Biochemistry. 2009 ; Vol. 48, No. 11. pp. 2459-2467.
@article{020c46f98583415c807cf8c19354a9d2,
title = "Crystal structure of acivicin-inhibited γ-glutamyltranspeptidase reveals critical roles for Its C-Terminus in autoprocessing and catalysis",
abstract = "Helicobacter pylori γ-glutamyltranspeptidase (HpGT) is a general γ-glutamyl hydrolase and a demonstrated virulence factor. The enzyme confers a growth advantage to the bacterium, providing essential amino acid precursors by initiating the degradation of extracellular glutathione and glutamine. HpGT is a member of the N-terminal nucleophile (Ntn) hydrolase superfamily and undergoes autoprocessing to generate the active form of the enzyme. Acivicin is a widely used γ-glutamyltranspeptidase inhibitor that covalently modifies the enzyme, but its precise mechanism of action remains unclear. The time-dependent inactivation of HpGT exhibits a hyperbolic dependence on acivicin concentration with k max = 0.033 ± 0.006 s- 1 and K I = 19.7 ± 7.2 μM. Structure determination of acivicin-modified HpGT (1.7 {\AA}; R factor = 17.9{\%}; R free = 20.8{\%}) demonstrates that acivicin is accommodated within the γ-glutamyl binding pocket of the enzyme. The hydroxyl group of Thr 380, the catalytic nucleophile in the autoprocessing and enzymatic reactions, displaces chloride from the acivicin ring to form the covalently linked complex. Within the acivicin-modified HpGT structure, the C-terminus of the protein becomes ordered with Phe 567 positioned over the active site. Substitution or deletion of Phe 567 leads to a > 10-fold reduction in enzymatic activity, underscoring its importance in catalysis. The mobile C-terminus is positioned by several electrostatic interactions within the C-terminal region, most notably a salt bridge between Arg 475 and Glu 566. Mutational analysis reveals that Arg 475 is critical for the proper placement of the C-terminal region, the Tyr 433 containing loop, and the proposed oxyanion hole.",
author = "Kristin Williams and Sierra Cullati and Aaron Sand and Biterova, {Ekaterina I.} and Barycki, {Joseph J.}",
year = "2009",
month = "3",
day = "24",
doi = "10.1021/bi8014955",
language = "English (US)",
volume = "48",
pages = "2459--2467",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "11",

}

TY - JOUR

T1 - Crystal structure of acivicin-inhibited γ-glutamyltranspeptidase reveals critical roles for Its C-Terminus in autoprocessing and catalysis

AU - Williams, Kristin

AU - Cullati, Sierra

AU - Sand, Aaron

AU - Biterova, Ekaterina I.

AU - Barycki, Joseph J.

PY - 2009/3/24

Y1 - 2009/3/24

N2 - Helicobacter pylori γ-glutamyltranspeptidase (HpGT) is a general γ-glutamyl hydrolase and a demonstrated virulence factor. The enzyme confers a growth advantage to the bacterium, providing essential amino acid precursors by initiating the degradation of extracellular glutathione and glutamine. HpGT is a member of the N-terminal nucleophile (Ntn) hydrolase superfamily and undergoes autoprocessing to generate the active form of the enzyme. Acivicin is a widely used γ-glutamyltranspeptidase inhibitor that covalently modifies the enzyme, but its precise mechanism of action remains unclear. The time-dependent inactivation of HpGT exhibits a hyperbolic dependence on acivicin concentration with k max = 0.033 ± 0.006 s- 1 and K I = 19.7 ± 7.2 μM. Structure determination of acivicin-modified HpGT (1.7 Å; R factor = 17.9%; R free = 20.8%) demonstrates that acivicin is accommodated within the γ-glutamyl binding pocket of the enzyme. The hydroxyl group of Thr 380, the catalytic nucleophile in the autoprocessing and enzymatic reactions, displaces chloride from the acivicin ring to form the covalently linked complex. Within the acivicin-modified HpGT structure, the C-terminus of the protein becomes ordered with Phe 567 positioned over the active site. Substitution or deletion of Phe 567 leads to a > 10-fold reduction in enzymatic activity, underscoring its importance in catalysis. The mobile C-terminus is positioned by several electrostatic interactions within the C-terminal region, most notably a salt bridge between Arg 475 and Glu 566. Mutational analysis reveals that Arg 475 is critical for the proper placement of the C-terminal region, the Tyr 433 containing loop, and the proposed oxyanion hole.

AB - Helicobacter pylori γ-glutamyltranspeptidase (HpGT) is a general γ-glutamyl hydrolase and a demonstrated virulence factor. The enzyme confers a growth advantage to the bacterium, providing essential amino acid precursors by initiating the degradation of extracellular glutathione and glutamine. HpGT is a member of the N-terminal nucleophile (Ntn) hydrolase superfamily and undergoes autoprocessing to generate the active form of the enzyme. Acivicin is a widely used γ-glutamyltranspeptidase inhibitor that covalently modifies the enzyme, but its precise mechanism of action remains unclear. The time-dependent inactivation of HpGT exhibits a hyperbolic dependence on acivicin concentration with k max = 0.033 ± 0.006 s- 1 and K I = 19.7 ± 7.2 μM. Structure determination of acivicin-modified HpGT (1.7 Å; R factor = 17.9%; R free = 20.8%) demonstrates that acivicin is accommodated within the γ-glutamyl binding pocket of the enzyme. The hydroxyl group of Thr 380, the catalytic nucleophile in the autoprocessing and enzymatic reactions, displaces chloride from the acivicin ring to form the covalently linked complex. Within the acivicin-modified HpGT structure, the C-terminus of the protein becomes ordered with Phe 567 positioned over the active site. Substitution or deletion of Phe 567 leads to a > 10-fold reduction in enzymatic activity, underscoring its importance in catalysis. The mobile C-terminus is positioned by several electrostatic interactions within the C-terminal region, most notably a salt bridge between Arg 475 and Glu 566. Mutational analysis reveals that Arg 475 is critical for the proper placement of the C-terminal region, the Tyr 433 containing loop, and the proposed oxyanion hole.

UR - http://www.scopus.com/inward/record.url?scp=64849086541&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=64849086541&partnerID=8YFLogxK

U2 - 10.1021/bi8014955

DO - 10.1021/bi8014955

M3 - Article

C2 - 19256527

AN - SCOPUS:64849086541

VL - 48

SP - 2459

EP - 2467

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 11

ER -