Intrinsic Activity and Stability of Bifunctional Human UMP Synthase and Its Two Separate Catalytic Domains, Orotate Phosphoribosyltransferase and Orotidine-5′-phosphate Decarboxylase

Michael J Yablonski,Daniel A Pasek,Byoung-Don Han,Mary Ellen Jones,Thomas W Traut

doi:10.1074/jbc.271.18.10704

Michael J Yablonski, Daniel A Pasek + Show 3 more

Open Access

https://doi.org/10.1074/jbc.271.18.10704

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Abstract

Human UMP synthase is a bifunctional protein containing two separate catalytic domains, orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine-5'-phosphate decarboxylase (EC 4.1.1.23). These studies address the question of why the last two reactions in pyrimidine nucleotide synthesis are catalyzed by a bifunctional enzyme in mammalian cells, but by two separate enzymes in microorganisms. From existing data on subunit associations of the respective enzymes and calculations showing the molar concentration of enzyme to be far lower in mammalian cells than in microorganisms, we hypothesize that the covalent union in UMP synthase stabilizes the domains containing the respective catalytic centers. Evidence supporting this hypothesis comes from studies of stability of enzyme activity in vitro, at physiological concentrations, of UMP synthase, the two isolated catalytic domains prepared by site-directed mutagenesis of UMP synthase, and the yeast ODCase. The two engineered 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.

Highlights

The final two steps in the de novo biosynthesis of UMP require the addition of ribose-P to the pyrimidine base orotate by orotate phosphoribosyltransferase (OPRTase)1 to form OMP and the subsequent decarboxylation of OMP to form UMP by orotidylate decarboxylase (ODCase)
These two catalytic centers are coded by two separate genes, while in all multicellular eukaryotes examined, the genes for these two catalytic centers have been joined into a single gene, resulting in the expression of the bifunctional protein, UMP synthase, with two different catalytic domains [1] (Fig. 1A)
An important feature of this model is that there are two conformational states of the dimer, based on sedimentation studies showing that only nucleotides produced the more rapidly sedimenting 5.6 S species [2, 3], on studies showing that a different type of tryptic digestion pattern for the pure enzyme was obtained in the presence of these tight binding nucleotides, than in the presence of simple anions [4], and on studies showing that enzyme preincubated to be in the 5.6 S form had optimum activity immediately after the addition of substrate, while enzyme preincubated in the 5.1 S form had a 20-s lag time after substrate addition before attaining optimum activity [5]

Summary

Monomeric UMP synthase can be converted to a simple

§ Present address: Central Research Institute, Hanil Pharmaceutical Co. Ltd., 656-408, 1 Ka 2 Dong, Sungsu Sungdong-Ku, Seoul, 133-122, Korea. There is better support for the other two benefits, since the dimerization of UMP synthase in response to effectors results in better activity for each catalytic domain as suggested in Fig. 1 [2, 3, 5, 9] Such an architecture could serve to enhance the stability of the enzyme as a monomer or as a dimer. This latter benefit was suggested by the observation described in the text that the intracellular concentrations of monofunctional OPRTase and ODCase in microorganisms are 10 –100-fold the concentration of the bifunctional UMP synthase in mammalian cells and was verified by the experiments described

EXPERIMENTAL PROCEDURES

Enzyme species

RESULTS AND DISCUSSION

Rat brain