Secondary 18O and primary 13C isotope effects as a probe of transition-state structure for enzymatic decarboxylation of oxalacetate.
A new method for directly measuring 18O isotope effects on decarboxylation reactions has been developed. By running the reaction under high vacuum (10(-5) torr), CO2 leaves the solution before exchange with the oxygens of water to an extent greater than 2%. Thus, the method permits determination of 18O isotope effects with the precision of the isotope ratio mass spectrometer, and without the necessity of resorting to the remote label method and its attendant required syntheses. The method is used to determine 18O isotope effects for decarboxylation of oxalacetate (OAA) by Mg2+, and enzymatically by OAA decarboxylase from Pseudomonas putida; 13C isotope effects are also reported for this enzyme, as well as for decarboxylation of OAA by pyruvate kinase. Initial velocity patterns and pH profiles are reported for the P. putida enzyme, and all available data are used to discuss the kinetic and chemical mechanism of decarboxylation.
Waldrop
G L
GL
Department of Biochemistry, College of Medicine, University of Tennessee, Memphis 38163.
Braxton
B F
BF
Urbauer
J L
JL
Cleland
W W
WW
Kiick
D M
DM
D002247:Carbon Isotopes; D002262:Carboxy-Lyases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007553:Isotope Labeling / Q000379:methods; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008433:Mathematics; D010071:Oxaloacetates / Q000737:chemistry* / Q000378:metabolism*; D010103:Oxygen Isotopes; D016958:Pseudomonas putida / Q000201:enzymology
D002247:Carbon Isotopes; D010071:Oxaloacetates; D010103:Oxygen Isotopes; D002262:Carboxy-Lyases; C021916:oxaloacetate decarboxylase; D008274:Magnesium