Effects of Sulfanilamide and Methotrexate on 13C Fluxes through the Glycine Decarboxylase/Serine Hydroxymethyltransferase Enzyme System in Arabidopsis1

In C3 plants large amounts of photorespiratory glycine (Gly) are converted to serine by the tetrahydrofolate (THF)-dependent activities of the Gly decarboxylase complex (GDC) and serine hydroxymethyltransferase (SHMT). Using 13C nuclear magnetic resonance, we monitored the flux of carbon through the GDC/SHMT enzyme system in Arabidopsis thaliana (L.) Heynh. Columbia exposed to inhibitors of THF-synthesizing enzymes. Plants exposed for 96 h to sulfanilamide, a dihydropteroate synthase inhibitor, showed little reduction in flux through GDC/SHMT. Two other sulfonamide analogs were tested with similar results, although all three analogs competitively inhibited the partially purified enzyme. However, methotrexate or aminopterin, which are confirmed inhibitors of Arabidopsis dihydrofolate reductase, decreased the flux through the GDC/SHMT system by 60% after 48 h and by 100% in 96 h. The uptake of [α-13C]Gly was not inhibited by either drug class. The specificity of methotrexate action was shown by the ability of 5-formyl-THF to restore flux through the GDC/SHMT pathway in methotrexate-inhibited plants. The experiments with sulfonamides strongly suggest that the mitochondrial THF pool has a long half-life. The studies with methotrexate support the additional, critical role of dihydrofolate reductase in recycling THF oxidized in thymidylate synthesis.

Cloning of human adenosine kinase cDNA: sequence similarity to microbial ribokinases and fructokinases.

Adenosine kinase catalyzes the phosphorylation of adenosine to AMP and hence is a potentially important regulator of extracellular adenosine concentrations. Despite extensive characterization of the kinetic properties of the enzyme, its primary structure has never been elucidated. Full-length cDNA clones encoding catalytically active adenosine kinase were obtained from lymphocyte, placental, and liver cDNA libraries. Corresponding mRNA species of 1.3 and 1.8 kb were noted on Northern blots of all tissues examined and were attributable to alternative polyadenylylation sites at the 3' end of the gene. The encoding protein consists of 345 amino acids with a calculated molecular size of 38.7 kDa and does not contain any sequence similarities to other well-characterized mammalian nucleoside kinases, setting it apart from this family of structurally and functionally related proteins. In contrast, two regions were identified with significant sequence identity to microbial ribokinase and fructokinases and a bacterial inosine/guanosine kinase. Thus, adenosine kinase is a structurally distinct mammalian nucleoside kinase that appears to be akin to sugar kinases of microbial origin.

Expression of human inducible nitric oxide synthase in a tetrahydrobiopterin (H4B)-deficient cell line: H4B promotes assembly of enzyme subunits into an active dimer.

Murine inducible nitric oxide (NO) synthase (iNOS) is catalytically active only in dimeric form. Assembly of its purified subunits into a dimer requires H4B. To understand the structure-activity relationships of human iNOS, we constitutively expressed recombinant human iNOS in NIH 3T3 cells by using a retroviral vector. These cells are deficient in de novo H4B biosynthesis and the role of H4B in the expression and assembly of active iNOS in an intact cell system could be studied. In the absence of added H4B, NO synthesis by the cells was minimal, whereas cells grown with supplemental H4B or the H4B precursor sepiapterin generated NO (74.1 and 63.3 nmol of nitrite per 10(6) cells per 24 h, respectively). NO synthesis correlated with an increase in intracellular H4B but no increase in iNOS protein. Instead, an increased percentage of dimeric iNOS was observed, rising from 20% in cytosols from unsupplemented cells to 66% in H4B-supplemented cell cytosols. In all cases, only dimeric iNOS displayed catalytic activity. Cytosols prepared from H4B-deficient cells exhibited little iNOS activity but acquired activity during a 60- to 120-min incubation with H4B, reaching final activities of 60-72 pmol of citrulline per mg of protein per min. Reconstitution of cytosolic NO synthesis activity was associated with conversion of monomers into dimeric iNOS during the incubation. Thus, human iNOS subunits dimerize to form an active enzyme, and H4B plays a critical role in promoting dimerization in intact cells. This reveals a post-translational mechanism by which intracellular H4B can regulate iNOS expression.

Interaction with the recombination hot spot chi in vivo converts the RecBCD enzyme of Escherichia coli into a chi-independent recombinase by inactivation of the RecD subunit.

The RecBCD enzyme of Escherichia coli promotes recombination preferentially at chi nucleotide sequences and has in vivo helicase and strong duplex DNA exonuclease (exoV) activities. The enzyme without the RecD subunit, as in a recD null mutant, promotes recombination efficiently but independently of chi and has no nucleolytic activity. Employing phage lambda red gam crosses, phage T4 2- survival measurements, and exoV assays, it is shown that E. coli cells in which RecBCD has extensive opportunity to interact with linear chi-containing DNA (produced by rolling circle replication of a plasmid with chi or by bleomycin-induced fragmentation of the cellular chromosome) acquire the phenotype of a recD mutant and maintain this for approximately 2 h. It is concluded that RecBCD is converted into RecBC during interaction with chi by irreversible inactivation of RecD. After conversion, the enzyme is released and initiates recombination on other DNA molecules in a chi-independent fashion. Overexpression of recD+ (from a plasmid) prevented the phenotypic change and providing RecD after the change restored chi-stimulated recombination. The observed recA+ dependence of the downregulation of exoV could explain the previously noted "reckless" DNA degradation of recA mutants. It is proposed that chi sites are regulatory elements for the RecBCD to RecBC switch and thereby function as cis- and trans-acting stimulators of RecBC-dependent recombination.

An allosteric switch controls the procoagulant and anticoagulant activities of thrombin.

Thrombin is an allosteric enzyme existing in two forms, slow and fast, that differ widely in their specificities toward synthetic and natural amide substrates. The two forms are significantly populated in vivo, and the allosteric equilibrium can be affected by the binding of effectors and natural substrates. The fast form is procoagulant because it cleaves fibrinogen with higher specificity; the slow form is anticoagulant because it cleaves protein C with higher specificity. Binding of thrombomodulin inhibits cleavage of fibrinogen by the fast form and promotes cleavage of protein C by the slow form. The allosteric properties of thrombin, which has targeted two distinct conformational states toward its two fundamental and competing roles in hemostasis, are paradigmatic of a molecular strategy that is likely to be exploited by other proteases in the blood coagulation cascade.