Pterins evidence

The molyhdopterin cofactor, as found in different enzymes, may be present either as the nucleoside monophosphate or in the dinucleotide form. In some cases the molybdenum atom binds one single cofactor molecule, while in others, two pterin cofactors coordinate the metal. Molyhdopterin cytosine dinucleotide (MCD) is found in AORs from sulfate reducers, and molyhdopterin adenine dinucleotide and molyb-dopterin hypoxanthine dinucleotide were reported for other enzymes (205). The first structural evidence for binding of the dithiolene group of the pterin tricyclic system to molybdenum was shown for the AOR from Pyrococcus furiosus and D. gigas (199). In the latter, one molyb-dopterin cytosine dinucleotide (MCD) is used for molybdenum ligation. Two molecules of MGD are present in the formate dehydrogenase and nitrate reductase. 

There are a number of studies on the biosynthesis of various pteridines, i.e., xanthopterin (65), isoxanthopterin (67), erythropterin (73), leucopterin (68), and pterin (62) (509-511). The most important intermediate of the proposed biosynthetic pathway from guanosine triphosphate (GTP) (604) seems to be di-hydroneopterin triphosphate (H2-NTP) (605), however, because evidence has recently been accumulated indicating that pteridines such as biopterin (70), sepiapterin (81), and drosopterins (87) are synthesized from GTP (604) by way of H2-NTP (605) (Scheme 76) (5/2). 

The importance of the dihydro and tetrahydro oxidation states of pterins in biology has stimulated interest in the study of the chemical properties of these compounds, especially with respect to electron-transfer and radical reactions. It has become apparent, perhaps unsurprisingly, that the stability and reactivity of these oxidation states are very sensitive to substituent effects and the much greater stability of the fully conjugated pteridines is most evident. The oxidation of tetrahydropterins and the reduction of dihydropterins have become especially important in the chemistry of nitric oxide production in nature and in oxidative stress but the accumulation of relevant facts has not led so far to a detailed understanding of the chemical property relationships. Relevant information is summarized in the following section. 

The expansion in the power of computers and theoretical methods has made it possible to investigate the mechanism of action of enzymes by combinations of quantum-mechanical and molecular-mechanical calculations. A study of two possible mechanisms for dihydrofolate reductase catalysis was consistent with indirect proton transfer from aspartate to N-5 of the pterin as has been suggested for many years by crystallographic evidence <2003PCB14036>. This conclusion is also consistent with the outcome of a study that directly measured the of the active site aspartate in the Lactobacillus casei enzyme <1999B8038>. Observations of chemical shifts of... 

The evidence for a pterin-substituted 1,2-enedithiolate was first reported by Raja-gopalan, Johnson, and coworkers, who isolated pterins from the oxidative decomposition of molybdenum-bound MPT, Figure 4 [7,49,55,56], In complementary work, Taylor and coworkers confirmed the structure of several of the pterin decomposition products by direct synthesis (see Section V. A) [30,57-59], Urothi-one, first isolated in 1940 from human urine [60], was shown to be a metabolic degradation product of MPT [37], Other isolated pterin-containing decomposition and/or derivatized products from molybdenum enzymes include Form A, Form B (a urothione-like product), and camMPT (Figure 4) [7], Two other pterins, Form Z and the MPT precursor, can be obtained from molybdenum deprived organisms, N. crassa Nit-1, and oxidase-deficient children, neither of which pro-... 

Stronger chemical evidence for the presence of a dithiolene in molybdopterin was obtained when the mild alkylation reagent iodoacetamide effectively trapped the dithiolene (65). This reaction yielded a derivative whose characterization by FAB mass spectrometry and nuclear magnetic resonance (NMR) was consistent with the structure shown in Fig. 5. The method appeared to leave the side chain intact and preserved the pterin oxidation state. From this experiment the view persisted that molybdopterin is a disubstituted dithiolene bearing a reduced pterin and a short chain terminated with a phosphate. 

While the evidence is undeniable for electron transfer via the pterin system for enzymes in the XO/XDH and AOR families, comparable structural features are not observed in SO. The additional electron-transfer group, the heme, is quite distant from the pterin ring system (Mo Fe 32 A) prohibiting an efficient electron transfer between these cofactors in the solid state. Because a flexible polypeptide chain connects the two domains housing the heme and the Moco, one postulation under investigation is that in solution the heme domain moves to position the heme closer to the pterin system to receive electrons during catalysis. 

Molybdopterin has another function besides participating in electron transfer between the site of catalysis and other electron-acceptor groups. It serves as an anchor for the active site where a multitude of hydrogen bonds between the pterin (and, if present, the dinucleotide) and the protein provide a secure tether for the reactive metal site (17). Evidence for the immobility conferred by the pterin(s) embedded in the protein is found in a comparsion of the DMSOR structures from both Rhodobacter sources. Regardless of the Mo coordination environment, the MGD ligands are nearly superimposable (75). This similarity of pterin structure is most clearly observed in the 1.3-A structure, where the Mo atom dissociated and shifted away from one pterin ligand, which otherwise was unaffected. The nucleotide tails on MGD, MCD, and other derivatives of molybdopterin also contribute to locking the molybdenum catalyst in position. 

Three human redox enzymes, and a variety of bacterial enzymes, contain molybdenum chelated by two sulfur atoms in a modified pterin molybdopterin (see Figure 10.1). In sulfite oxidase, the other two chelation sites of the molybdenum are occupied by oxygen in xanthine oxidase / dehydrogenase (Section 7.3.7) and aldehyde oxidase, one site is occupied by oxygen and one by sulfur. In some bacterial enzymes, molybdopterin occurs as a guanine dinucleotide rather than free. In others, tungsten rather than molybdopterin is the chelated metal there is no evidence that any mammalian enzymes contain tungsten. 

Molybdopterin itself is also extremely unstable when released from a protein and has never been structurally characterized in its native state (32, 33). Mass spectral and NMR studies of the difcarboxamido-methyl) derivative of the oxidized form of molybdopterin have provided convincing evidence that this derivative is a 6-substituted pterin that possesses structure 3 (34). A 6-substituted pterin moiety now appears to be a common feature of all of the molybdenum enzymes of Table I. There is still some question about the oxidation state of the pterin ring... 

In summary, a 6-substituted pterin was first identified as a structural component of the molybdenum cofactor from sulfite oxidase, xanthine oxidase and nitrate reductase in 1980 (24). Subsequent studies provided good evidence that these enzymes possessed the same unstable molyb-dopterin (1), and it seemed likely that 1 was a constituent of all of the enzymes of Table I. It now appears that there is a family of closely related 6-substituted pterins that may differ in the oxidation state of the pterin ring, the stereochemistry of the dihydropterin ring, the tautomeric form of the side chain, and the presence and nature of a dinucleotide in the side chain. In some ways the variations that are being discovered for the pterin units of molybdenum enzymes are beginning to parallel the known complexity of naturally occurring porphyrins, which may have several possible side chains, various isomers of such side chains, and a partially reduced porphyrin skeleton (46). 

Evidence for the presence of a pterin cofactor in this enzyme was obtained by Mukund and Adams (282) the fluorescence spectrum of the... 

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