Metabolic studies rate-limiting step

Experimental studies of Methanosarcina and current understanding of the organism s metabolic pathway allow us to estimate the parameters in the thermodynamic term (Qusheng Jin, personal communication). The methanogens conserve about 24 kJ (mol acetate)-1, so we set AGp to 48 kJ mol-1 and m to one half. A double proton translocation occurs within the central metabolic pathway, furthermore, so, if we take these as the rate limiting steps, the average stoichiometric number / is two. 

Although demethylation, which occurs in the liver, is normally considered to be a catabolic process, it may result in conversion of an inactive form of a drug to the active form. Thus 6-(methylthio)purine (XXXIX) is demethylated by the rat to 6-mercaptopurine [205]. This demethylation occurs in the liver micro-somes and is an oxidative process which converts the methyl group to formaldehyde [204, 207]. The 1-methyl derivative of 4-aminopyrazolo[3,4-d] pyrimidine (XLI) is demethylated slowly, but 6-mercapto-9-methylpurine (XLII) not at all [208]. The A -demethylation of puromycin (XLlIl) [209, 210], its aminonucleoside (XLIV) [211], and a number of related compounds, including V-methyladenine and V,V-dimethyladenine, occurs in the liver microsomes of rodents [212]. In the guinea-pig the rate-limiting step in the metabolism of the aminonucleoside appears to be the demethylation of the monomethyl compound, which is the major urinary metabolite [213]. The relationship of lipid solubility to microsomal metabolism [214], and the induction of these demethylases in rats by pre-treatment with various drugs have been studied [215]. 

The enzymes involved in the polyamine metabolic pathway have been the subject of intensive study, and a number of specific inhibitors for these enzymes have been designed as potential antitumor or antiparasitic agents [166]. Thus, a-difluoromethylornithine, has become a clinically useful agent [167]. Most of the studies involving inhibitors of polyamine metabolism have focused on enzymes involved in the biosynthetic pathway. Recently, there has been considerable interest generated in the enzyme spermidine/spermine-hT -acetyltrans-ferase enzyme (SSAT), the rate-limiting step in the back conversion of polyamines. SSAT, in conjunction with polyamine oxidase (PAO), allows for reversal of the biosynthetic pathway and attenuation of the levels of individual polyamines. 

The manner in which the reduction of ribonucleotides to deoxyribonucleotides is regulated has been studied with reductases from relatively few species. The enzymes from E. coli and from Novikoff s rat liver tumor have a complex pattern of inhibition and activation (fig. 23.25). ATP activates the reduction of both CDP and UDP. As dTTP is formed by metabolism of both dCDP and dUDP, it activates GDP reduction, and as dGTP accumulates, it activates ADP reduction. Finally, accumulation of dATP causes inhibition of the reduction of all substrates. This regulation is reinforced by dGTP inhibition of the reduction of GDP, UDP, and CDP and by dTTP inhibition of the reduction of the pyrimidine substrates. Because evidence suggests that ribonucleotide reductase may be the rate-limiting step in deoxyribonucleotide synthesis in at least some animal cells, these allosteric effects may be important in controlling deoxyribonucleotide synthesis. 

Very little information is available on the mechanisms that control the production of PE via the CDP-ethanolamine pathway. Factors that regulate this pathway for PE production at the level of gene expression have not yet been elucidated. In the 1970s Akesson and Sundler reported that ET catalyzes the rate-limiting step of this pathway (R. Sundler, 1975). In addition, under some metabolic conditions the supply of the substrate, DG, can limit the rate of PE biosynthesis from ethanolamine (L.M.G. van Golde, 1989). Two studies have implicated a channeling of intermediates of the pathway in the biosynthesis of PE in mammalian cells (M.W. Spence, 1989). 

There are conflicting data on whether the availability of cholesterol and/or cholesteryl esters directly influences apo B secretion. Several studies have suggested that cholesterol supply can regulate VLDL secretion. For example, VLDL production in animals and man is decreased by statin treatment, and inhibition of cholesterol synthesis by a statin, an inhibitor of the rate-limiting step of cholesterol biosynthesis (Chapter 14), reduced VLDL secretion in perfused rat livers (M. Heimberg, 1990) and primary hepatocytes. However, this effect of statins can perhaps be ascribed to increased expression of LDL receptors rather than to a reduction in cholesterol synthesis (Section 7.1). Depletion of cholesterol in rodent hepatocytes by the ABCAl-dependent lipidation of apo A1 (Chapter 19) also decreases VLDL secretion (R. Lehner, 2004). Furthermore, the secretion of apo BlOO-containing VLDLs is increased in primary hepatocytes derived from Niemann-Pick Cl-deficient mice. Niemann-Pick Cl-deficiency causes a severe defect in trafficking of unesterified cholesterol out of the lysosomal/endosomal pathway and consequently, Niemann-Pick Cl-deficient hepatocytes accumulate 5- to 10-fold more unesterified cholesterol than do wild-type hepatocytes. In hepatocytes from Niemann-Pick Cl-deficient mice, cholesterol synthesis is increased and the rate of cholesterol esterification and the amount of the transcriptionally active form of SREBP-1 are also increased (J.E. Vance, 2007). However, because of multiple alterations in lipid metabolism in these hepatocytes, increased VLDL secretion cannot be attributed specifically to increased synthesis of cholesterol or cholesteryl esters. 

Heme, the most abundant iron cofactor, can play diversified roles in the cell. These roles include not only the already-mentioned regulatory and signal transduction processes, but also electron transfer, oxygen binding and transport, and direct involvement in the oxygen metabolism. The first step of the heme biosynthetic pathway in mammalian cells is catalyzed by 5-aminolevulinic acid synthase (ALAS), which is considered a rate-limiting step in the production of heme. The rate of synthesis of erythroid ALAS is directly dependent on the cellular iron concentration. Ferreira reviews recent structural and site-directed mutagenesis studies on ALAS (Chapter 2), which, for example, have revealed that the homodimeric enzyme s active site is located at the subunit interface and contains catalytically essential residues from both subunits. 

A similar correlation approach (but not as a cocktail ) was used to elucidate similar rate-limiting steps in the metabolism of dihydropyridine calcium channel blockers. Racemic felodipine, racemic nitrendipine and nifedipine were investigated in a randomized cross-over study in healthy subjects, using stereoselective enantiomers. The high correlations between the AUCs of all compounds strongly suggest the involvement of the same or very similar enzyme(s) in their primary oxidative metabolism, which in all cases involves aromatization of the dihydropyridine ring stmcture [22]. 

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