The 18O isotope effect in 13C nuclear magnetic resonance spectroscopy: mechanistic studies on asparaginase from Escherichia coli.
The mechanism of the enzyme asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) from Escherichia coli was examined using 13C NMR spectroscopy. The pH-dependent oxygen exchange reactions between water and aspartic acid were followed by use of the 18O isotope-induced shift of the resonance positions of directly bonded 13C nuclei. Both L-1- and L-1,4-[13C]aspartic acid were used in experiments with previously 18O-labeled aspartic acid, or in experiments involving the use of 18O-labeled solvent water. Asparaginase catalyzes a relatively efficient exchange between the oxygens of water and those on one carboxyl group of aspartic acid. Exchange at C-4 occurs rapidly but, within experimental error, no exchange at C-1 could be detected. These and related experiments involving the position of 18O incorporation during hydrolysis of aspartic acid beta-methyl ester are all consistent with possible acyl-enzyme mechanisms involving C-4, but do not support a free aspartic acid anhydride mechanism.
D001215:Asparaginase / Q000378:metabolism*; D001224:Aspartic Acid / Q000378:metabolism; D002247:Carbon Isotopes; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010103:Oxygen Isotopes; D014867:Water / Q000378:metabolism
D002247:Carbon Isotopes; D010103:Oxygen Isotopes; D014867:Water; D001224:Aspartic Acid; D001215:Asparaginase