Iso-Orotate Decarboxylase: Attemps at purification by mechanism based affinity chromatography, /

Abstract

Iso-orotate decarboxylase (IDCase) has been partially purified by affinity chromatography. The protein lysates used in this purification were from Neurospora crassa. Affinity chromatography was carried out by synthesizing N-3-(3-carboxypropyl) 5-nitrouracil (N3 conjugated 5NU) as the ligand. Partial purification was successful yielding a 7-fold purification scheme. Kinetic and inhibition data was collected for IDCase from Rhodotorula glutinis. The Km, Michaelis constant, ofIDCase was determined to be 64.45 ± 0.05 !J.M. IDCase from N crassa had a Km of35 ± 9 !J.M, meaning that IDCase from R. glutinis has less affinity for the substrate, iso-orotate (IDA), than the enzyme from N crassa. The Kj for 5NU, a potent inhibitor ofIDCase, was found to be -25 nM while the inhibitor had a Kj of-0.5 nM from N crassa IDCase. This indicated that R. glutinis IDCase has a lower affinity for 5NU than N crassa IDCase. The Kj for N3 conjugated 5NU for IDCase from R. glutinis was established as -7.9 !J.M, which suggests that IDCase binds to this inhibitor with more affinity than the substrate. Since IDCase binds to N3 conjugated 5NU with high affinity, the compound could be used as a ligand in affinity chromatography. The proposed mechanism of IDCase involves a covalent attachment to C6 of IDA. This was somewhat demonstrated by an experiment in which labeled inhibitor-was added to IDCase and the protein was trapped on a membrane. The results show that inhibitor alone does not bind to the membrane while IDCase with the inhibitor does.