INTRINSIC ACTIVITY AND STABILITY OF BIFUNCTIONAL HUMAN UMP SYNTHASE AND ITS 2 SEPARATE CATALYTIC DOMAINS, OROTATE PHOSPHORIBOSYLTRANSFERASEE AND OROTIDINE-5'-PHOSPHATE DECARBOXYLASE

Human UMP synthase is a bifunctional protein containing two separate c atalytic domains, orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine-5'-phosphate decarboxylase (EC 4.1.1.23), These studies addr ess the question of why the last two reactions in pyrimidine nucleotid e synthesis are catalyzed by a bi-functional enzyme in mammalian cells , but by two separate enzymes in microorganisms, From existing data on subunit associations of the respective enzymes and calculations showi ng the molar concentration of enzyme to be far lower in mammalian cell s than in microorganisms, we hypothesize that the covalent union in UM P synthase stabilizes the domains containing the respective catalytic centers, Evidence supporting this hypothesis comes from studies of sta bility of enzyme activity in vitro, at physiological concentrations, o f UMP synthase, the two isolated catalytic domains prepared by site-di rected mutagenesis of UMP synthase, and the yeast ODCase, The two engi neered domains have activities very similar to the native UMP synthase , but unlike the bifunctional protein, the domains are quite unstable under conditions promoting the dissociated monomer.