Formation and isolation of a covalent intermediate during the glutaminase reaction of a class II amidotransferase.
Incubation of Escherichia coli asparagine synthetase B (AS-B) with [14C]-L-glutamine gives a covalent adduct that can be isolated. Radiolabeled protein is not observed (i) when the wild-type enzyme is incubated with 6-diazo-5-oxo-L-norleucine (DON) prior to reaction with [14C]glutamine or (ii) when the C1A AS-B mutant is incubated with [14C]-L-glutamine. Both of these alterations eliminate the ability of the enzyme to utilize glutamine but do not affect ammonia-dependent asparagine synthesis. Formation of the covalent adduct therefore depends on the presence of the N-terminal active site cysteine, which has been shown to be essential for glutamine-dependent activity in this and other class II amidotransferases. The amount of covalent adduct exhibits saturation behavior with increasing concentrations of L-glutamine. The maximum observed quantity of this intermediate is consistent with its involvement on the main pathway of glutamine hydrolysis. The chemical properties of the isolable covalent adduct are consistent with those anticipated for the gamma-glutamyl thioester that has been proposed as an intermediate in the AS-B-catalyzed conversion of glutamine to glutamate. The covalent adduct is acid-stable but is labile under alkaline conditions. On the basis of the measured rates of formation and breakdown of this intermediate, it is kinetically competent to participate in the normal catalytic mechanism. These studies represent the first description of a thioester intermediate for any class II amidotransferase and represent an important step in gaining further insight into the kinetic and chemical mechanisms of AS-B.
Schnizer
H G
HG
Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, Florida 32610, USA.
Boehlein
S K
SK
Stewart
J D
JD
Richards
N G
NG
Schuster
S M
SM
D001217:Aspartate-Ammonia Ligase / Q000737:chemistry* / Q000378:metabolism; D002250:Carbon Radioisotopes; D004926:Escherichia coli / Q000201:enzymology; D005972:Glutaminase / Q000737:chemistry* / Q000378:metabolism; D005973:Glutamine / Q000737:chemistry* / Q000378:metabolism; D019811:Hydroxylamine / Q000378:metabolism; D007700:Kinetics
D002250:Carbon Radioisotopes; D005973:Glutamine; D019811:Hydroxylamine; D005972:Glutaminase; D001217:Aspartate-Ammonia Ligase