Iso-orotate decarboxylase: Measurement of kinetic constants for substrates and inhibitors, and attempted gene isolation, by Julie M. Angelot.

Abstract

Iso-orotate decarboxylase (IDCase) activity has been measured in protein lysates and ammonium sulfate fractionated suspensions prepared from wild type and mutant strains of Neurospora crassa using 14COz-displacement assays and UV-vis spectrophotometric assays. The activity of IDCase toward an alternate substrate, 2-thio-iso-orotate, which has an absorbance maximum outside the range of that for total protein, has been observed. The Michaelis-Menten constant for the original substrate, IDA, and alternate substrate, TIOA, have been measured to be Km -35 J-LM +/- 9 and Km -224 J-LM +/- 53 respectively. 5-nitrouracil is a strong inhibitor of IDCase activity, with a K too low for measurement by enzyme inhibition studies. Inhibtion of 5NU has been theoretically estimated to be -0.5 nM. 5-nitrocytosine is a much less potent inhibitor, Kj -26 J-LM. The Kj of its tautomeric form was also theoretically estimated to be -2.5 nM giving rise to the possibility that the tautomer is responsible for inhibition activity towards IDCase. The inhibition and alternate substrate data is interpreted in a model for IDCase catalysis, in which a nucleophilic enzyme group makes a covalent attachment to the substrate prior to decarboxylation. Purification using dye ligands as affinity chromatography resisns have yielded a definite negative result. IDCase did not preferentially bind to any column resisn tested. Gene isolation experiments using bacterial complementation techniques have yielded a negative result.