Ribotide, formation

Since ribose phosphate compounds are involved at some stage in purine synthesis, knowledge of the mechanism of ribotide formation has become a fundamental aspect of the purine problem. The exact enzymatic step mediating the coupling of ribose phosphate to a purine precursor is not known, although the addition is thought to occur before the AIC skeleton is completed (in view of the negative bank experiments discussed above). Since AIC and hypoxanthine are both converted to their respe< -tive ribotides, it was hoped that a study of hypoxanthine conversion to inosinic acid would reveal some of the important aspects of the fundamental reactions of ribotide synthesis. 

The pathways for thiamine biosynthesis have been elucidated only partiy. Thiamine pyrophosphate is made universally from the precursors 4-amino-5-hydroxymethyl-2-methylpytimidinepyrophosphate [841-01-0] (47) and 4-methyl-5-(2-hydroxyethyl)thiazolephosphate [3269-79-2] (48), but there appear to be different pathways ia the eadier steps. In bacteria, the early steps of the pyrimidine biosynthesis are same as those of purine nucleotide biosynthesis, 5-Aminoimidazole ribotide [41535-66-4] (AIR) (49) appears to be the sole and last common iatermediate ultimately the elements are suppHed by glycine, formate, and ribose. AIR is rearranged in a complex manner to the pyrimidine by an as-yet undetermined mechanism. In yeasts, the pathway to the pyrimidine is less well understood and maybe different (74—83) (Fig. 9). 

So far, we have constructed an unsatisfying picture of the earliest days of an RNA world although some prebiotic mechanisms may exist for the untemplated formation of oligonucleotides, these molecules would have been short, would have contained a variety of monomers besides ribotides, and could not have been faithfully copied by the template-directed polymerization of monomers. Given this model, it is difficult to imagine the accumulation of RNA sequences necessary for the Darwinian selection of a multitude of active ribozymes. Nevertheless, these precursors may have been adequate for the first critical step in the formation of life the formation of an RNA replicase. 

Tryptophan is an essential amino acid involved in synthesis of several important compounds. Nicotinic acid (amide), a vitamin required in the synthesis of NAD+ and NADP+, can be synthesized from tryptophan (Figure 17-24). About 60 mg of tryptophan can give rise to 1 mg of nicotinamide. The synthesis begins with conversion of tryptophan to N-formylkynurenine by tryptophan pyrrolase, an inducible iron-porphyrin enzyme of liver. N-Formylkynurenine is converted to kynurenine by removal of formate, which enters the one-carbon pool. Kynurenine is hydroxylated to 3-hydroxykynurenine, which is converted to 3-hydroxyanthranilate, catalyzed by kynureninase, a pyridoxal phosphate-dependent enzyme. 3-Hydroxyanthranilate is then converted by a series of reactions to nicotinamide ribotide, the immedi-... 

In order to carry out GC-MS analysis of cytokinins, which are mainly N -substituted derivatives of adenine that occur in plants as free bases, ribosides, ribotides and glucosides, it is necessary to convert these polar compounds into volatile derivatives. There are, however, technical problems as the more commonly used t-BuDMS and TMS derivatives are partially and completely hydrolysed, respectively, in aqueous solvents and therefore cannot be purified by reverse phase HPLC [110]. Multiple derivative formation is also known to occur during trimethylsilylation [111]. Permethyl derivatives are stable in aqueous solutions but the preparation of reagents and the derivatization procedures are both time consuming and complex and, in addition, formation of multiple derivatives can also occur [6]. Frit-FAB HPLC-MS of cytokinins is therefore particularly useful as standards and plant extracts can be analysed without recourse to derivatization [101]. Tabulated positive ion FAB mass spectra of a range of cytokinins are presented in Table... 

To date, no biochemical evidence exists to support this notion of a bifunctional role for adenylosuccinate synthetase. In particular, the synthesis of specific enzymes has not been correlated with the loss of regulation. Instead, the loss of regulation has been followed by measuring purine excretion or by observing adenine-insensitive accumulation and polymerization of aminoimidazole ribotide (red color formation) in cells blocked at adel or add2. In addition, adenylosuccinate synthetase from S. cerevisiae has not been purified or characterized and the... 

Amino-4-imidazole carboxamide ribotide, a precursor only two steps removed (formylation and cycli-zation) from inosinic acid, can be synthesized by the direct condensation of the imidazole with 5-phosphori-bosyl pyrophosphate. The enzyme catalyzing this reaction was purified from an acetone powder of beef liver. The same enzyme (AMP pyrophosphorylase) catalyzes the condensation of adenine, guanine, and hypoxan-thine. Nucleoside phosphorylase is an enzyme that catalyzes the formation of a ribose nucleoside from a purine base and ribose-1-phosphate. Guanine, adenine, xanthine, hypoxanthine, 2,6-diaminopurine, and aminoimidazole carboxamide are known to be converted to their respective nucleosides by such a mechanism. In the presence of a specific kinase and ATP, the nucleoside is then phosphorylated to the corresponding nucleotide. 

Comparing the excretion patterns of allopurinol-1-riboside and orotic acid/orotidine in the urine over a period of 24 hours after allopurinol administration(subject 4, fig. 2) we conclude from our data that allopurinol-1-ribotide plays only a minor role in oroticaciduria(day 2,3). Additional evidence provided the administration of hypoxanthine 5 hours after allopurinol(day 4) The excretion of allopurinol-1-riboside was not influenced whereas the excretion of orotic acid was reduced to the same extent as on day 5 and 6. On these days hypoxanthine was given together with allopurinol and inhibited the formation of allopurinol-1-riboside completely. 

The inhibitory effect of allopurinol on the first three steps of purine biosynthesis in Neurospora crassa cultures could be due 1. to the formation of allopurin-ol-l-ribonucleotide (by HGPRTase 1,19 nMol/mg protein/ hr in our Neurospora crassa cultures) 2. due to an oxipurinol-7-ribonucleotide effect, namely the formation of oxipurinol (by xanthine oxidase) and then oxipurinol-7-ribonucleotide (by OPRTase) and 3. by formation of allopurinol-1-ribotide through allopurin-ol-1-riboside (by a purine nucleoside phosphorylase) to allopurinol-1-ribonucleotide (by a nucleoside kinase). This latter pathway seems rather improbable because we could not demonstrate an effect on purine biosynthesis by direct addition of allopurinol-1-ribonucleoside to the incubation medium. Allopurinol-1-ribonucleotide inhibits significantly at higher concentrations. This is to explain if there is free access of the ribonucleotide form through the cell membrane (at least in these high concentrations) or if there is conversion to the ribonucleoside (which then enters the cell) by a nucleotidase located on the cell membrane. This latter mechanism however seems more improbable bcause there was no effect by allopurinol-1-ribonucleoside (which excludes the possibility for subsequent formation of allopurinol-1 -ribonucleotide by a nucleoside kinase, at least... 

Role of 4-Amino-B-imidazolecarhoxamide (A/C) and Its Ribotide. When C -AIC was incubated with pigeon Uver homogenates or extracts in the presence of a-ketoglutarate, there was an appreciable utilization of the compound which could be accounted for by the formation of radioactive hypoxanthine. The formation of isotopic hypoxanthine from HC OOH was stimulated by the addition of nonlabeled AIC in amounts indicating that labeled formate and AIC react approximately in eriui-molar proportion in the synthesis. ... 

In order to explain the enzymatic exchange reaction, it was assumed that inosinic acid was enzymatically degraded to 4-amino-5-imi-dazolecarboxamide ribotide, and that the latter compound reacted vith radioactive formate, yielding inosinic acid labeled in the 2-position. It was further postulated that the citrovorum factor was the coenzjune of... 

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