Formation of IMP

In mammals specific enzymes for converting purine bases to nucleotides are present in many organs, and in heart muscle this may be the main source of purine nucleotides. The most important of these enzymes is hypoxanthine-guanine phosphoribosyltransferase, which catalyzes the formation of IMP from hypoxanthine and GMP from guanine ... 

By far the most common method of formation of IMPs is the Staudinger reaction.1 This is the reduction of an organic azide with triphenylphosphine (Scheme 4). It has been proven that attack of the phosphorus is at the terminal nitrogen of... 

The application of the HPLC assay method to studies on reaction mechanisms has been limited, and the reader is referred to the work of Sloan (1984). Sloan and his colleagues studied the formation of IMP or GMP (and pyrophosphate) from the substrates phosphoribosylpyrophosphate (PRPP) and either hypoxanthine or guanine. These reactions, catalyzed by hypoxan-thine/guanine phosphoribosyltransferase (GHPRTase), were studied by HPLC after a method was developed to separate all the reactants and products simultaneously. 

The strategy used was as follows. First the initial rates of IMP and GMP formation were studied separately, and from an inspection of the doublereciprocal plots obtained from these data, it was determined that product formation proceeds by a sequential kinetic mechanism. Next the formation of IMP and GMP was studied for several concentrations of both bases. Finally, the rate of formation of the common product, pyrophosphate, was examined over a series of fixed ratios of hypoxanthine to guanine. 

Hypoxanthine guanosine phosphoribosyltransferase (HGPRT) catalyzes the formation of IMP and pyrophosphate (PPj) from hypoxanthine (Hyp) and phosphoribosylpyrophosphate (PRibPP) as shown in reaction (1) ... 

Figure 9.98 HPLC elution profiles of an incubation mixture to study hypoxanthine/ guanine phosphoribosyltransferase. The reaction was initiated by the addition of the enzyme mixture, and aliquots were injected onto the HPLC column at 10-minute intervals as indicated on the z axis. The solid peaks represent hypoxanthine, which decreases with time, while the hatched peaks describe the formation of IMP. (From Jahngen and Rossomando, 1984.)...

The results of an experiment are shown in Figure 10.6. The formation of GMP and IMP from guanine and hypoxanthine, respectively, can be followed. With this method it was possible to track the initial rates of formation of IMP and GMP separately, the initial rates of both determined simultaneously, and the rate of PRibPP utilization with a fixed ratio of hypoxanthine and guanine. 

Figure 10.7 Schematic representation of the formation and fate of IMP. Formation of IMP is catalyzed by the enzyme hypoxanthine/guanine phosphoribosyl tranfer-ase (1) from the substrate hypoxanthine (Hypo) and phosphoribosyl pyrophosphate (PRibPP). IMP is shown undergoing several reactions the first (2) is catalyzed by 5 -nucleotidase to form inosine (INO) and orthophosphate (Pj) the other (3) is a two-step reaction catalyzed by sAMP synthetase to form adenylosuccinate (sAMP) and (4) by the enzyme sAMP lyase to convert sAMP to AMP and fumarate. Finally, (3) the deamination of AMP to IMP and NHa is catalyzed by AMP deaminase.

Ammonia is produced by oxidative and nonoxidative deaminations catalyzed by glutaminase and glutamate dehydrogenase (Chapter 17). Ammonia is also released in the purine nucleotide cycle. This cycle is prominent in skeletal muscle and kidney. Aspartate formed via transamination donates its a-amino group in the formation of AMP the amino group is released as ammonia by the formation of IMP. 

The formation of IMP may provide a means by which the intracellular purine nucleotide pool is maintained. AMP deaminase deficiency disrupts the purine... 

The degree of dissociation, x of the micelles was determined from the specific conductance vs. concentration of surfactants plot. Actually, x is the ratio of the post micellar slope to the premicellar slope of these plots. The counter ion association, y of the micelles is equal to (1 -x). The results of cmc and y values obtained for IMP micelles in absence and presence of KCl at different temperatures are given in Table 4. It is found that the cmc of IMP in aqueous solution increased with increase in temperature, whereas the cmc of IMP decreased in the presence of additive (KCl) at all temperatures mentioned above (see Table 4). The increase in cmc and decrease in y values for IMP micelles in aqueous solution suggest that the micelle formation of IMP is hindered with the increase in temperature. However, the micelle formation of IMP is more facilitated in the presence of KQ even at higher temperatures showing lower cmc and higher y values (see Table 4). 

Antioxidants. The following active inhibitors may be added separately or in combination to the fuel in total concentration not in excess of 8.4 pounds of inhibitor (not including weight of solvent) per 1000 barrels of fuel (9.1gm/l00 US gal, 24mg/liter or 109 mg/imp gal) in order to prevent the formation of gum ... 

Three major feedback mechanisms cooperate in regulating the overall rate of de novo purine nucleotide synthesis and the relative rates of formation of the two end products, adenylate and guanylate (Fig. 22-35). The first mechanism is exerted on the first reaction that is unique to purine synthesis—transfer of an amino group to PRPP to form 5-phosphoribosylamine. This reaction is catalyzed by the allosteric enzyme glutamine-PRPP amidotransferase, which is inhibited by the end products IMP, AMP, and GMP. AMP and GMP act synergisti-cally in this concerted inhibition. Thus, whenever either AMP or GMP accumulates to excess, the first step in its biosynthesis from PRPP is partially inhibited. 

In the second control mechanism, exerted at a later stage, an excess of GMP in the cell inhibits formation of xanthylate from inosinate by IMP dehydrogenase, without affecting the formation of AMP (Fig. 22-35). Conversely, an accumulation of adenylate inhibits formation of adenylosuccinate by adenylosuccinate synthetase, without affecting the biosynthesis of GMP. In the third mechanism, GTP is required in the conversion of IMP to AMP (Fig. 22-34, step (T)), whereas ATP is required for conversion of IMP to GMP (step (4)), a reciprocal arrangement that tends to balance the synthesis of the two ribonucleotides. 

Summary of incorporation of precursors into the purine ring of IMP. In steps 3 and 9 formate or other indirect donors of a one-carbon unit, such as serine, donate their atoms in the form of a formyl group attached to reduced folate. 

Conversion of IMP to AMP and GMP. In both cases two steps are required. Note that the formation of AMP requires GTP, and the formation of GMP requires ATP. This tends to balance the flow of the IMP down the two pathways. 

Next, in steps 7 and 8, N-l of the purine ring is contributed by aspartate. Aspartate forms an amide with the 4-carboxyl group, and the succinocarboxamide so formed is then cleaved with release of fumarate. Energy for carboxamide formation is provided by ATP hydrolysis to ADP and phosphate. These reactions resemble the conversion of cit-rulline to arginine in the urea cycle (chapter 22) and the conversion of IMP to AMP (see fig. 23.11). 

All biosynthetic pathways are under regulatory control by key allosteric enzymes that are influenced by the end products of the pathways. For example, the first step in the pathway for purine biosynthesis is inhibited in a concerted fashion by nucleotides of either adenine or guanine. In addition, the nucleoside monophosphate of each of these bases inhibits its own formation from inosine monophosphate (IMP). On the other hand, adenine nucleotides stimulate the conversion of IMP into GMP, and GTP is needed for AMP formation. 

Looking at compounds with direct taste effects, the significance of amino acids and nucleotides in the formation of potato taste has been described in several papers (3,4,5). The free amino acids and 5 -nucleotides are certainly an important fraction they contribute to taste due to their content of glutamic acid, aspartic acid, 5 -AMP, 5 -IMP and other compounds. From the vast literature two analytical examples which have also been tested in taste tests are presented in Table II. 

Daptomycin localises to the cell division septum in B. subtilis 34 it causes rapid cell death without lysis,48 causes the formation of aberrant cell wall septa48 and treats biofilms effectively in S. aureus. 9 Daptomycin induces the cell wall stress stimulon in S. aureus and B. subtilis,30 34 but has very poor activity (MIC of 128) against an E. coli imp mutant defective in outer membrane assembly,5 whereas vancomycin, another bulky peptide that normally does not penetrate the outer membrane, has an MIC of 0.8 against E. coli imp.50... 

The most widespread use of IMPs is in the aza-Wittig reaction. This is the reaction of an IMP with a carbonyl group to generate an imine, or derivative thereof, with concomitant formation of the corresponding phosphine oxide (Scheme 2). The reaction is successful with a wide range of carbonyl containing compounds such as aldehydes, ketones, acyl chlorides, amides, and in some cases esters.2 Exposure to carbon dioxide, carbon disulfide, isocyanates, isothiocyanates, and... 

The final example is slightly different from the previous protocols in two ways. First, the IMP is generated by Sn2 displacement of a benzotriazole moiety. The IMP then opens an epoxide, which generates a betaine with an extra carbon atom between phosphorus and the negatively charged oxygen. Collapse of this betaine results in the formation of an aziridine as opposed to a carbon-nitrogen double bond. 

The possibility that more of the reaction product Ado had remained on the column was ruled out by using different mobile phases to elute all bound material. The chromatograms accounted for all the products. Since we had not expected any side reactions, we quantitated the yield of reaction products on the basis of area, assuming the presence of only adenosine-containing compounds. The formation of inosine, with a 50% reduction in extinction coefficient, could account for the apparent lack of recovery. Therefore, we considered the presence of secondary reactions. Either AMP had been converted to IMP, or adenosine was converted to inosine (Ino). By comparing the retention time of the third peak to authentic standards, we ruled out IMP as a product, and thus the identity of peak 3 was established as inosine. This led us to conclude that the commercial preparation of alkaline phosphatase was contaminated with a second activity, adenosine deaminase. 

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