Mechanistic inferences from the crystal structure of fumarylacetoacetate hydrolase with a bound phosphorus-based inhibitor.

Fumarylacetoacetate hydrolase (FAH) catalyzes the hydrolytic cleavage of a carbon-carbon bond in fumarylacetoacetate to yield fumarate and acetoacetate as the final step of Phe and Tyr degradation. This unusual reaction is an essential human metabolic function, with loss of FAH activity causing the fatal metabolic disease hereditary tyrosinemia type I (HT1). An enzymatic mechanism involving a catalytic metal ion, a Glu/His catalytic dyad, and a charged oxyanion hole was previously proposed based on recently determined FAH crystal structures. Here we report the development and characterization of an FAH inhibitor, 4-(hydroxymethylphosphinoyl)-3-oxo-butanoic acid (HMPOBA), that competes with the physiological substrate with a K(i) of 85 microM. The crystal structure of FAH complexed with HMPOBA refined at 1.3-A resolution reveals the molecular basis for the competitive inhibition, supports the proposed formation of a tetrahedral alkoxy transition state intermediate during the FAH catalyzed reaction, and reveals a Mg(2+) bound in the enzyme's active site. The analysis of FAH structures corresponding to different catalytic states reveals significant active site side-chain motions that may also be related to catalytic function. Thus, these results advance the understanding of an essential catabolic reaction associated with a fatal metabolic disease and provide insight into the structure-based development of FAH inhibitors.

Cloning, overproduction, and purification of native and mutant recombinant yeast orotate phosphoribosyltransferase and the demonstration from magnetization inversion transfer that a proposed oxocarbocation intermediate does not have a kinetic lifetime.

The gene for orotate phosphoribosyltransferase from Saccharomyces cerevisiae has been subcloned into an Escherichia coli overexpression vector and the enzyme has been produced in large quantities, thus simplifying the purification to one step. We were able to repeat the published (J. Victor, L. B. Greenberg, and D. L. Sloan J. Biol. Chem. 254, 2647-2655, 1979). 32PPi/5-phosphorylribose 1-alpha-diphosphate exchange experiments and could demonstrate the exchange by magnetization inversion transfer NMR experiments as well. However, when contaminating orotidine 5'-monophosphate (OMP) was eliminated with OMP decarboxylase, any evidence of magnetization transfer vanished. Consequently, it is concluded that a ping pong mechanism is not operable and that a previously proposed oxocarbocation intermediate along the pathway to OMP does not persist long enough in the catalytic cycle of this enzyme to be recognized by NMR exchange experiments.

Does the bifunctional uridylate synthase channel orotidine 5'-phosphate? Kinetics of orotate phosphoribosyltransferase and orotidylate decarboxylase activities fit a noninteracting sites model.

Uridylate synthase is a bifunctional protein that first forms orotidine 5'-phosphate (OMP) from orotate via its orotate phosphoribosyltransferase activity (EC 2.4.2.10) and then converts OMP to uridine 5'-phosphate (UMP) via the OMP decarboxylase activity (EC 4.1.1.23). A computer modeling analysis of the experiments that led to the proposal [Traut, T.W., & Jones, M.E. (1977) J. Biol. Chem. 252, 8374-8381] that uridylate synthase channels intermediate OMP suggests that the experimental results do not demonstrate preferential use of OMP generated in the bifunctional complex as against exogenous OMP. This analysis shows that the experimentally observed amounts of [6-14C]UMP from [6-14C]orotate in the presence of various amounts of exogenous [7-14C]OMP agree well with the amounts predicted by the computer simulations. Thus we conclude that uridylate synthase does not channel OMP. Additionally, the subsequent suggestion that channeling of OMP occurs to protect the intermediate from degradation by a nucleotidase [Traut, T.W. (1980) Arch. Biochem. Biophys. 200, 590-594] seems unlikely. The appropriate computer simulation demonstrates that low transient levels of OMP and protection of the intermediate are provided for strictly by the kinetic parameters of orotate phosphoribosyltransferase, OMP decarboxylase, and the nucleotidase. Additionally, calculations show that, in both sets of published experiments, the concentration of transient OMP greatly exceeded the concentration of OMP decarboxylase active sites. Thus, channeling of OMP by the bifunctional complex cannot be invoked to explain the evolution of uridylate synthase, and that event must be the result of some other selective pressure.

Inhibition of fructose bisphosphatase and stimulation of phosphofructokinase by a stable isosteric phosphonate analog of fructose 2,6-bisphosphate.

We describe the synthesis of a mixture of D-manno- and D-gluco-2,5-anhydro-1-deoxy-1-phosphonohexitol 6-phosphate via a Horner-Emmons reaction of 2,3,5-tri-O-benzyl-beta-D-arabinofuranose followed by phosphorylation of the equivalent 6-position and subsequent deprotection. This mixture inhibits fructose-1,6-bisphosphatase; the concentration required for half-maximal effect in the presence of 25 microM AMP is approximately 6 microM. The mixture of analogs also stimulates 6-phosphofructo-1-kinase from rabbit liver; the concentration required to reach one-half Vmax was found to be ca. 25 microM at 0.25 mM fructose 6-phosphate and 50 microM AMP. These analogs have replaced the labile anomeric phosphate of fructose 2,6-bisphosphate with a stable methylenephosphonate, and could be of great interest due to their appropriate physiological effects and their chemical stability.

Potential for ambiguity in the interpretation of immunochemical studies on multienzymic proteins. The case of uridylate synthase.

Rabbit antibodies directed against homogeneous uridylate synthase multienzyme from mouse Ehrlich ascites carcinoma precipitate both the orotidine-5'-monophosphate decarboxylase (EC 4.1.1.23) and orotate phosphoribosyltransferase (EC 2.4.2.10) activities of mouse and human erythrocyte uridylate synthase. When the partially purified human enzyme is used as antigen the two activities coprecipitate with the same apparent titer; however, when the mouse carcinoma protein was studied under the same conditions the decarboxylase activity immunoprecipitated with significantly higher avidity than did the transferase activity. Since the mouse multienzyme has been shown to be a single polypeptide that contains both activities (McClard, R.W., Black, M.J., Livingstone, L.R. and Jones, M.E. (1980) Biochemistry 19, 4699-4706), these results were, at face value, surprising. However, when the mouse orotate phosphoribosyltransferase activity (which is largely lost upon dilution into the immunoassay medium) was stabilized with 5-phosphoribosyl 1-pyrophosphate, both enzyme activities displayed the same apparent antibody titer. The immunochemical studies indicate that the antibodies, as a population, preferentially bind to a form or forms of the enzyme which contain(s) denatured transferase domains. A calculation based on a simple model yields a value of approximately 100 for the relative selectivity of the antibody for the denatured form of uridylate synthase. These results illustrate an ambiguity that is inherent in the interpretation of immunochemical studies on such multienzymic proteins; that is, it is possible to conclude incorrectly that two enzyme activities are not functionally associated if one of the catalytic domains is particularly unstable and thereby displays greater immunoreactivity for the specific antiserum.

Rapid determination of anti-enzyme titer in serum using wholeStaphylococcus aureus cells.

Heat-killed, formalin-fixedStaphyloccous aureus (Cowan) cells are shown to be an excellent immunoadsorbent for quickly and accurately measuring the titer of antibodies directed against enzymes. The simple procedure is illustrated by following the course of immunization of a rabbit against mouse uridine 5't -monophosphate synthase. The antibody titer of a serum sample can be determined in roughly 1 h plus the time to perform the assays of enzyme (antigen) activity.

Isolation and initial characterization of the single polypeptide that synthesizes uridine 5'-monophosphate from orotate in Ehrlich ascites carcinoma. Purification by tandem affinity chromatography of uridine-5'-monophosphate synthase.

UMP synthase, or multienzyme pyr-5,6 (orotate phosphoribosyltransferase:orotidine monophosphate decarboxylase), has been purified from Ehrlich ascites carcinoma to apparent homogeneity. The purification was achieved by the use of 5-[2-[N-(2-aminoethyl)carbamyl]ethyl]-6-azauridine 5'-monophosphate-agarose and phosphocellulose affinity columns linked in tandem by a flow dialysis system. The purified protein has amolecular weight of approximately 51500 as judged by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Both enzyme activities cosediment with an S20,w value of 3.7 S, which corresponds to a molecular weight of about 50000. Two-dimensional electrophoresis of UMP synthase shows that the protein exists as two isomeric forms with isoelectric points of 5.85 (major form) and 5.65 (minor form). Both forms have the same molecular weight of 51500 and contain both active centers. These results clearly show that the last two enzyme activities of de novo UMP biosynthesis occur on a single polypeptide chain of approximately 51500 daltons and that this polypeptide exists in at least two isomeric forms.