PMID- 10527932
OWN - NLM
STAT- MEDLINE
DCOM- 20000103
LR  - 20181113
IS  - 0264-6021 (Print)
IS  - 0264-6021 (Linking)
VI  - 343 Pt 3
DP  - 1999 Nov 1
TI  - Site-directed mutagenesis establishes cysteine-110 as essential for enzyme
      activity in human gamma-glutamyl hydrolase.
PG  - 551-5
AB  - Gamma-glutamyl hydrolase (GH), which hydrolyses the gamma-glutamyl conjugates of 
      folic acid, is a key enzyme in the maintenance of cellular folylpolyglutamate
      concentrations. The catalytic mechanism of GH is not known. Consistent with
      earlier reports that GH is sulphydryl-sensitive, we found that recombinant human 
      GH is inhibited by iodoacetic acid, suggesting that at least one cysteine is
      important for activity [Rhee, Lindau-Shepard, Chave, Galivan and Ryan (1998) Mol.
      Pharmacol. 53, 1040-1046]. Using site-directed mutagenesis, the cDNA for human GH
      was altered to encode four different proteins each with one of four cysteine
      residues changed to alanine. Three of the mutant proteins had activities similar 
      to wild-type GH and were inhibited by iodoacetic acid, whereas the C110A mutant
      had no activity. Cys-110 is conserved among the human, rat and mouse GH amino
      acid sequences. The wild-type protein and all four mutants had similar intrinsic 
      fluorescence spectra, indicating no major structural changes had been introduced.
      These results indicate that Cys-110 is essential for enzyme activity and suggest 
      that GH is a cysteine peptidase. These studies represent the first identification
      of the essential Cys residue in this enzyme and provide the beginning of a
      framework to determine the catalytic mechanism, important in defining GH as a
      therapeutic target.
FAU - Chave, K J
AU  - Chave KJ
AD  - Division of Molecular Medicine, Wadsworth Center, New York State Department of
      Health, Albany, NY 12201-0509, USA.
FAU - Galivan, J
AU  - Galivan J
FAU - Ryan, T J
AU  - Ryan TJ
LA  - eng
GR  - CA25933/CA/NCI NIH HHS/United States
PT  - Journal Article
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - England
TA  - Biochem J
JT  - The Biochemical journal
JID - 2984726R
RN  - 0 (DNA Primers)
RN  - 0 (Recombinant Proteins)
RN  - EC 3.4.19.9 (gamma-Glutamyl Hydrolase)
RN  - K848JZ4886 (Cysteine)
RN  - WF5188V710 (Iodoacetic Acid)
SB  - IM
MH  - Amino Acid Sequence
MH  - Amino Acid Substitution
MH  - Animals
MH  - Binding Sites
MH  - Conserved Sequence
MH  - *Cysteine
MH  - DNA Primers
MH  - Humans
MH  - Iodoacetic Acid/pharmacology
MH  - Kinetics
MH  - Mice
MH  - Molecular Sequence Data
MH  - Mutagenesis, Site-Directed
MH  - Rats
MH  - Recombinant Proteins/antagonists & inhibitors/chemistry/metabolism
MH  - Sequence Alignment
MH  - Sequence Homology, Amino Acid
MH  - gamma-Glutamyl Hydrolase/antagonists & inhibitors/*chemistry/*metabolism
PMC - PMC1220585
EDAT- 1999/10/21 00:00
MHDA- 1999/10/21 00:01
CRDT- 1999/10/21 00:00
PHST- 1999/10/21 00:00 [pubmed]
PHST- 1999/10/21 00:01 [medline]
PHST- 1999/10/21 00:00 [entrez]
PST - ppublish
SO  - Biochem J. 1999 Nov 1;343 Pt 3:551-5.