The mechanism of the adenylosuccinate synthetase reaction as studied by positional isotope exchange

In an attempt to gain insight into the mechanism of the rat muscle adenylosuccinate synthetase reaction, experiments using the technique of positional isotope exchange (isotope scrambling) were undertaken. [gamma-18O]GTP was prepared and incubated with Mg2+ and the synthetase in the presence of vari...

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Bibliographic Details
Published in:The Journal of biological chemistry 1984-10, Vol.259 (20), p.12330-12333
Main Authors: Bass, M B, Fromm, H J, Rudolph, F B
Format: Article
Language:English
Subjects:
Adenylosuccinate Synthase - metabolism
Analytical, structural and metabolic biochemistry
Animals
Biological and medical sciences
Enzymes and enzyme inhibitors
Fundamental and applied biological sciences. Psychology
Guanosine Triphosphate - metabolism
Isotope Labeling - methods
Kinetics
Ligases
Ligases - metabolism
Magnesium
Muscles - enzymology
Oxygen Isotopes
Protein Binding
Rats
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Summary:In an attempt to gain insight into the mechanism of the rat muscle adenylosuccinate synthetase reaction, experiments using the technique of positional isotope exchange (isotope scrambling) were undertaken. [gamma-18O]GTP was prepared and incubated with Mg2+ and the synthetase in the presence of various ligands. Positional isotope exchange occurred, as measured by nuclear magnetic resonance spectroscopy, when IMP was present. In the absence of IMP, with or without aspartate or succinate, the [gamma-18O]GTP did not exhibit scrambling. These results suggest that the adenylosuccinate synthetase reaction involves the participation of 6-phosphoryl-IMP as an obligatory intermediate. On the basis of experiments carried out in our laboratory as well as in others, we believe the GDP remains bound to the enzyme until the product, adenylosuccinate, is formed. All products may then dissociate randomly from the enzyme. The positional isotope exchange experiments, along with initial-rate experiments carried out in our laboratory, serve to explain the lack of partial exchange reactions associated with the synthetase (Fromm, H. J. (1958) Biochim. Biophys. Acta 29, 255-262), as well as the net inversion of configuration when chiral thio-GTP is converted to thiophosphate (Webb, M. R., Reed, G. H., Cooper, B. F., and Rudolph, F. B. (1984) J. Biol. Chem. 259, 3044-3046).
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(18)90748-9