Elucidating the mechanism of action of orotidine monophosphate decarboxylase (ODCase) using isotope labeling, enzyme kinetics and 6-CN-UMP inhibitor

Abstract

Orotidine 5-Monophophatedecarboxylase (ODCase) is involved in the de novo biosynthesis of Uridine 5-Monophosohate (UMP) via a decarboxylation reaction using Orotidine 5-monophosphate as the substrate. UMP is used in the synthesis of pryimidines which are important components of the nucleotides. ODCase is one of the most proficient enzymes, however little is known about its mechanism of action. We are concerned with ascertaining the validity of the proposed. ODCase mechanism of action via a zwitterionic intermediate followed by subsequent decarboxylation at C6. By synthesizing a double labeled substrate(OMP) with the 13C labeled carbon at the carbonyl carbon at position 6 (the key position) and 18O labeled at position 2 (indicator position), the reaction is run to partial completion using the E. coli system. A separate experiment is run for the naturally occurring OMP. Bycarrying out isotope effect calculations we can ascertain whether O2 is involved in the ODCase mechanism of action or not [1]. According to Masahiro Fujihashi[2] when ODCase from M. thermoautotrophiciium was incubated with 6-CN-UMP showed diminished activity due to the formation of BMP a potent inhibitor. We are concerned in finding out if the same happens with ODCase from E. coli and whether the oxygen in BMP comes from water or dissolved oxygen.