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 [C-14]-L -glutamine gives a covalent adduct that can be isolated. Radiolabeled prote in is not observed (i) when the wild-type enzyme is incubated with 6-diazo- 5-oxo-L-norleucine (DON) prior to reaction with [C-14]glutamine or (ii) whe n the C1A AS-B mutant is incubated with [C-14]-L-glutamine. Both of these a lterations eliminate the ability of the enzyme to utilize glutamine but do not affect ammonia-dependent asparagine synthesis. Formation of the covalen t adduct therefore depends on the presence of the N-terminal active site cy steine, which has been shown to be essential for glutamine-dependent activi ty in this and other class II amidotransferases. The amount of covalent add uct exhibits saturation behavior with increasing concentrations of L-glutam ine. The maximum observed quantity of this intermediate is consistent with its involvement on the main pathway of glutamine hydrolysis. The chemical p roperties of the isolable covalent adduct are consistent with those anticip ated for the gamma-glutamyl thioester that has been proposed as an intermed iate in the AS-B-catalyzed conversion of glutamine to glutamate. The covale nt adduct is acid-stable but is labile under alkaline conditions. On the ba sis of the measured rates of formation and breakdown of this intermediate, it is kinetically competent to participate in the normal catalytic mechanis m. These studies represent the first description of a thioester intermediat e for any class II amidotransferase and represent an important step in gain ing further insight into the kinetic and chemical mechanisms of AS-B.