Probing the mechanism of nitrogen transfer in Escherichia coli asparagine synthetase by using heavy atom isotope effects.
In experiments aimed at determining the mechanism of nitrogen transfer in purF amidotransferase enzymes, 13C and 15N kinetic isotope effects have been measured for both of the glutamine-dependent activities of Escherichia coli asparagine synthetase B (AS-B). For the glutaminase reaction catalyzed by AS-B at pH 8.0, substitution heavy atom labels in the side chain amide of the substrate yields observed values of 1.0245 and 1.0095 for the amide carbon and amide nitrogen isotope effects, respectively. In the glutamine-dependent synthesis of asparagine at pH 8.0, the amide carbon and amide nitrogen isotope effects have values of 1.0231 and 1.0222, respectively. We interpret these results to mean that nitrogen transfer does not proceed by the formation of free ammonia in the active site of the enzyme and probably involves a series of intermediates in which glutamine becomes covalently attached to aspartate. While a number of mechanisms are consistent with the observed isotope effects, a likely reaction pathway involves reaction of an oxyanion with beta-aspartyl-AMP. This yields an intermediate in which C-N bond cleavage gives an acylthioenzyme and a second tetrahedral intermediate. Loss of AMP from the latter gives asparagine. An alternate reaction mechanism in which asparagine is generated from an imide intermediate also appears consistent with the observed kinetic isotope effects.
Stoker
P W
PW
Department of Biochemistry, Beadle Center, University of Nebraska-Lincoln, 68583, USA.
O'Leary
M H
MH
Boehlein
S K
SK
Schuster
S M
SM
Richards
N G
NG
D001217:Aspartate-Ammonia Ligase / Q000378:metabolism*; D001224:Aspartic Acid / Q000378:metabolism; D002247:Carbon Isotopes; D004926:Escherichia coli / Q000201:enzymology*; D005798:Genes, Bacterial; D005972:Glutaminase / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism; D007553:Isotope Labeling; D007700:Kinetics; D008956:Models, Chemical; D009587:Nitrogen Isotopes; D013379:Substrate Specificity
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D005973:Glutamine; D001224:Aspartic Acid; D005972:Glutaminase; D001217:Aspartate-Ammonia Ligase