PubMed Health⌕ Search
Home/Archive/PubMed

PubMed · 6698284

Human GMP synthetase.

T Page·B Bakay·W L Nyhan
View original source ↗Export metadata ↓

Abstract

The specific activity of GMP synthetase was measured in several human tissues and found to be highest in cultured skin fibroblasts, followed by bone marrow, leukocytes, erythrocytes, placenta, and liver. The enzyme from fibroblasts was purified approximately 50-fold by ammonium sulfate fractionation and gel filtration. The Km values were determined to be 4.9 microM for XMP, 270 microM for ATP, and 340 microM for glutamine. Ammonium sulfate could replace glutamine as the amino donor but was much less efficient. The enzyme was specific for ATP as the energy source. Unlike the calf thymus enzyme, the human enzyme has no requirement for a reduced sulfhydryl compound. Human GMP synthetase is inhibited by ATP, dATP, azaserine, and hydroxylamine.

Explore related subjects

Carbon-Nitrogen LigasesFibroblastsHumansIn Vitro TechniquesKineticsLigasesSkinTissue DistributionComparative StudyJournal ArticleResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, P.H.S.

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

T Page, B Bakay, W L Nyhan. 1984. Human GMP synthetase.. https://doi.org/10.1016/0020-711x(84)90061-2

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2006

The alternative pathway of glutathione degradation is mediated by a novel protein complex involving three new genes in Saccharomyces cerevisiae.

Glutathione (GSH), L-gamma-glutamyl-L-cysteinyl-glycine, is the major low-molecular-weight thiol compound present in almost all eukaryotic cells. GSH degradation proceeds through the gamma-glutamyl cycle that is initiated, in all organisms, by the action of gamma-glutamyl transpeptidase. A novel pathway for the degradation of GSH that requires the participation of three previously uncharacterized genes is described in the yeast Saccharomyces cerevisiae. These genes have been named DUG1 (YFR044c), DUG2 (YBR281c), and DUG3 (YNL191w) (defective in utilization of glutathione). Although dipeptides and tripeptides with a normal peptide bond such as cys-gly or glu-cys-gly required the presence of only a functional DUG1 gene that encoded a protein belonging to the M20A metallohydrolase family, the presence of an unusual peptide bond such as in the dipeptide, gamma-glu-cys, or in GSH, required the participation of the DUG2 and DUG3 gene products as well. The DUG2 gene encodes a protein with a peptidase domain and a large WD40 repeat region, while the DUG3 gene encoded a protein with a glutamine amidotransferase domain. The Dug1p, Dug2p, and Dug3p proteins were found to form a degradosomal complex through Dug1p-Dug2p and Dug2p-Dug3p interactions. A model is proposed for the functioning of the Dug1p/Dug2p/Dug3p proteins as a specific GSH degradosomal complex.

Carbon-Nitrogen Ligases↗
PubMed2006

Functional characterization of acetylglutamate synthase and phosphoribosylamine-glycine ligase genes in Gibberella zeae.

Gibberella zeae (anamorph, Fusarium graminearum) is an important pathogen of cereal crops found in many regions of the world. In this study, we have characterized two auxotrophic strains, designated S4B1279 and S4B3008, which were discovered from a collection of insertional mutants of G. zeae generated by restriction enzyme-mediated integration (REMI). Both mutant strains exhibited pleiotropic phenotypic changes that include reduction of mycelial growth and virulence and abolished sexual reproduction. Molecular analysis of the REMI mutants has shown that the auxotrophy of S4B1279 is due to a mutation of the ARG2 gene encoding an acetylglutamate synthase, and the auxotrophy of S4B3008 is due to a mutation of the ADE5 gene encoding a phosphoribosylamine-glycine ligase. Subsequent gene disruption and complementation studies have confirmed the functions for ARG2 and ADE5, respectively, in G. zeae. Our study has demonstrated the feasibility of using the REMI technique in studying G. zeae virulence mechanisms, in addition to providing two new selectable markers allowing genetic transformation of the fungus.

Carbon-Nitrogen Ligases↗
PubMed2006

Direct detection and kinetic analysis of covalent intermediate formation in the 4-amino-4-deoxychorismate synthase catalyzed reaction.

Chorismate-utilizing enzymes catalyze diverse reactions, providing critical physiological functions unique to plants, bacteria, fungi, and some parasites. Their absence in animals makes them excellent targets for antimicrobials and herbicides. 4-Amino-4-deoxychorismate synthase (ADCS) catalyzes the first step in folate biosynthsis and shares a common core mechanism with isochorismate synthase (IS) and anthranilate synthase (AS), in which nucleophile addition at C2 initiates these reactions. Evidence was presented previously [He, Z., Stigers Lavoie, K. D., Bartlett, P. A., and Toney, M. D. (2004) J. Am. Chem. Soc. 126, 2378-2385] that K274 is the nucleophile in ADCS, implying formation of a covalent intermediate. Herein, we report the direct detection of this covalent intermediate formed in ADCS-catalyzed reactions by ESI-MS. Difference spectra show the covalent intermediate has an absorption maximum at 310 nm. This was used to study the pre-steady-state kinetics of covalent intermediate formation under various conditions. Additionally, E258 in ADCS was shown to be critical to formation of the covalent intermediate by acting as a general acid catalyst for loss of the C4 hydroxyl group. The E258A/D mutants both exhibit very low activity. Acetate is a poor chemical rescue agent for E258D but an excellent one for E258A, with a 20000-fold and 3000-fold rate increase for Gln-dependent and NH(4)(+)-dependent activities, respectively. Lastly, A213 in IS (structurally homologous to K274 in ADCS) was changed to lysine in an attempt to convert IS to an ADCS-like enzyme. HPLC studies support the formation of a covalent intermediate with this mutant.

Carbon-Nitrogen Ligases↗