Partial purification and characterization of orotidine 5'-monophosphate decarboxylase in Escherichia coli : determination of biphasic kinetic parameters

Abstract

Orotidine 5'-monophosphate decarboxylase has been partially purified approximately 200-fold from Escherichia coli. The molecular weight determined by gel filtration was 56,000. The enzyme exhibited biphasic kinetics. A new method is presented for analyzing biphasic kinetic data from two independent saturable reactions assuming the lines to be tangents at average points for each of the two data regions. Km's so determined were 0.27 and 4.65 μM. The best inhibitors were 6-azauridine 5'-monophosphate > 8-azauridine 5'-monophosphate > xanthosine 5'-monophosphate > UMP > AMP. The pH optimum for 5 μM orotidine 5'-monophosphate was 7.0. An orotic acid excreting E. coli K-12 strain W3110 trpA33 had an orotidine 5 '-monophosphate decarboxylase which had nearly identical inhibition properties to an E. coli B 23 wild-type strain.