Methyltransferase donor substrate

Excess NAm in cell cultures is metabolized primarily by enzymatic methylation, catalyzed by nicotinamide methyltransferase, forming 1-methylnicotinamide (1-MNA), the primary excretory form (19, 20). This leads to depletion of cellular S-adenosyhnediionine (AdoMet), the methyl donor substrate, and an accumulation of S-adenosylhomocysteine (AdoHcy), a potent feedback inhibitor of enzymatic methylation reactions (21). Cellular levels of AdoMet and AdoHcy were measured as previously described (22). When FELCs were incubated wifli 20 mM NAm for four days, the AdoMet levels declined to almost half diat of control cells by 72 hr, with a concommitant increase in AdoHcy concentrations, which peaked at 48 hr (Fig. 3). The decline in AdoHcy levels after this time seemed to follow the return of DNA mediylation status by 96 hr, as determined earlier. Thus, excess cellular NAm levels result in a depletion of AdoMet and an increase in AdoHcy, indirectly causing hypomethylation of the DNA. 

As an overall conclusion it may be stated that the chain length as well as the amino and/or the carboxyl residue of the amino acid moiety appear to be critical in the binding of competitive inhibitors to the enzyme molecule. This confirms that the methyl donor substrate, Ado-Met, most likely binds through several of its chemical groups to the enzyme protein, as it has been previously indicated with other methyltransferase enzymes... 

Histone-lysine methyltransferases are chromatin-bound enzymes that catalyses the addition of methyl groups onto lysine or arginine residues of chromatin-bound H3 and H4 [151]. The methyl group is transferred enzymatically to the histone with S-adenosyl methionine as the methyl donor. Histone methylases have been isolated from HeLa S-3 cells [182], chick embryo nuclei [183], and rat brain chromatin [184]. The histone methyltransferases methylated H3 and H4 in nucleosomes [184]. Histone-lysine methyltransferase is a chromatin-bound enzyme [129,151]. Initial characterization of the Tetrahymena macronuclear H3 methyltransferase suggests that the enzyme has a molecular mass of 400 kDa. The enzyme preferred free histones rather than nucleosomes as substrate [138]. More recent studies have now... 

Fig. 1.43. The methylation of DNA 5-methyl-cytidine and maintenance methylation. a) The methylation of cytidine residues on DNA is catalyzed by a methyl transferase that employs S-ade-nosine methionine as a methyl group donor. The peferable substrate for the methyl transferase are hemi-methylated CpG sequences. 5-aza-cytidine is a specific inhibitor of methyl transferses. b) The methylation pattern of DNA remains intact upon DNA replication and is passed on to the daughter cells. The newly synthesized strands are unmethylated immediately after DNA rephca-tion. The methyltransferase uses the previously methylated parent strand as a matrix to methylate the CpG sequences of the newly synthesized strand.

Since preliminary studies showed that 6-hydroxymellein-O-methyl-transferase activity was appreciably inhibited in the presence of the reaction products, the mode of product inhibition of the enzyme was studied in detail in order to understand the regulatory mechanism of in vivo methyltransfer. It is well known that S-adenosyl-Z.-homocysteine (SAH), which is a common product of many O-methyltransferases that use SAM as methyl donor, is usually a potent inhibitor of such enzymes. In the 6-hydroxymellein-Omethyltransferase catalyzing reaction another product of this enzyme, 6-methoxymellein, has pronounced inhibitory activity, in addition to SAH. Since the specific product of the transferase reaction, 6-methoxymellein, is capable of inhibiting transferase activity [88], this observation suggests that activity of the transferase is specifically regulated in response to increases in cellular concentrations of its reaction products in carrot cells. It has been also found that 6-methoxymellein inhibits transferase activity with respect not only to 6-hydroxymellein but also to SAM, competitively. This competitive inhibition was also found in SAH as a function of the co-substrates of the enzyme [89]. It follows that the reaction catalyzed by 6-hydroxymellein-O-methyltransferase proceeds by a sequential bireactant mechanism in which the entry of the co-substrates to form the enzyme-substrate complexes and the release of the co-products to generate free enzyme take place in random order [Fig. (7)]. This result also implies that 6-methoxymellein and SAH have to associate with the free transferase protein to exhibit their inhibitory activities, and cannot work as the inhibitors after the enzyme forms complexes with the the substrate. If, therefore, 6-hydroxymellein-O-methyltransferase activity is controlled in vivo by its specific product 6-methoxymellein, this compound should... 

Another structural feature often found in NRPS products is N-methylated amide bonds. The domain that introduces this Ci unit, the so-called methyltransferase (Mt) domain, is situated between the A and the PCP domain (21). By consumption of 5-Adenosyl-methionine, the a-amino group of the acceptor substrate is methylated before condensation with the donor. 

Hydroxyl, as well as amino and thiol groups, may be metabolized through methylation, the methyl donor being S -adenosyl methionine, the product of the interaction of ATP with methionine. Usually, it is a minor metabolic route in xenobiotic metabolism, but it plays a major role in the metabolism of endogenous substrates such as noradrenaline (norepinephrine). Methylation is catalyzed by methyltransferases located in the mitochondria. 

MT family of enzymes catalyzes the O-, S-, or N-methylation of drugs, hormones, and neurotransmitters, and utilizes S-adenosyl-L-methionine (SAM) as a methyl donor. Catechol-O-methyltransferase (COMT) is the most extensively investigated drug-metabolizing MT. It plays an important role in the biotransformation of both endogenous and exogenous catechols. COMT has a rather broad substrate specificity for structures that contain catechol moieties and is often involved in the methylation of... 

Protein ( histidine ) Methyltransferase. An enzyme which methylates histidine in proteins to give primarily 3-methylhistidine residues has been observed in myofibrillar protein and in the sarcoplasmic fraction of muscle homogenates (218). S-Adenosyl-L-methionine serves as the methyl donor for the enzyme. The enzyme has not been solubilized and purified. Very little is known about the substrate specificity of protein-(histidine) methyltransferase. Actins from a wide variety of species consistently contain one 3-N-methylhistidine residue per molecule (191, 219). It appears that myosin from white muscle contains two residues of 3-N-methylhistidine (one residue per heavy chain), whereas myosin from red muscle contains no 3-N-methylhistidine (220). The amino acid sequence around the methylated residue of rabbit skeletal muscle is (221) ... 

The three steps of methylation of norlaudanosoline (iii, iv, v) are catalyzed by three kinds of methyltransferases (60MT, CNMT, and 4 OMT). These enzymes need 5-adenosyl-L-methionine (SAM) as the methyl donor. The 60MT and CNMT from C. japonica have broad substrate selectivity. Therefore, 60MT can catalyze the 6-(9-methlyation of not only norcoclaurine (VI) but also norlaudanosoline (iii). Similarly, CNMT can catalyze the N-methylation of not only coclaurine (VII) but also 6-(9-methylnorlaudanosoline (iv). 

Not only is S-adenosylethionine inactive as an ethyl donor for transfer RNA (Ortwerth and Novelli, 1969), but this ethyl analog also inhibits the methylation of transfer RNA by S-adenosylmethionine (Moore and Smith, 1969). The impression that S-adenosylethionine is a rather poor substrate for many methyltransferases is supported by the observation... 

Of the substrates for the betaine and thetin methyltransferases, only betaine is formed in the liver. Dimethylpropiothetin and S-methylme-thionine also occur naturally, but are formed only in plants and algae (Cantoni, 1960). It appears that, physiologically, betaine is the chief methyl donor for these enzymes in mammalian tissues with, perhaps, minor utilization of such S-methylmethionine or thetins as may be ingested in foods derived from plant material. Betaine is formed by oxidation of choline. Choline is formed by a series of transmethylations, each of which utilizes a molecule of S-adenosylmethionine. Clearly, therefore, the betaine-dependent pathway does not provide the animal with a means of... 

Catechol-O-methyltransferase from rat liver requires S-adeno-sylmethionine as a methyl donor and can methylate catechol but not monohydroxy derivatives of phenylethylamine. In vivo, O-methylation occurs exclusively in the meta position to the carbon side chain, but with purified preparations of the enzyme in vitro methylation can lead to both meta and para methylation. The ratio of products is susceptible. to both the polarity of the substrate and the pH of the medium . In man 3-methoxy-4-hydroxy-mandelic acid (18) comprises about 40 per cent of the total urinary metabolites produced from the catecholamines whilst in other pecies 3-methoxy-4-hydroxyphenylglycol (19), isolated as a sulphate ester, is the predominant breakdown product . A typical metabolic grid which indicates the possible types of pathway leading from noradrenalin (17) to both of these metabolites is shown in Figure 4.4 analogous metabolic schemes may be drawn up for both adrenalin and dopamine. 

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