Enzyme transmethylation

As part of a theoretical examination of the factors controlling the catalytic efficiency of a transmethylation enzyme (catechol (9-mcthyltransferase), the reaction mechanism of the non-enzymic transmethylation of catechol by, -adcnosylmethionine (AdoMet, as modelled by sulfonium ion) has been elucidated by using ab initio and semiempirical quantum mechanical methods.97 The gas-phase reaction between catecholate and sulfonium is extremely fast, involving no overall barrier, and the reaction profile to some extent resembles that of a typical gas-phase, S N 2 reaction. However, in aqueous solution, this reaction is very slow, with a predicted barrier of 37.3 kealmol-1. Good agreement between calculated KIEs for the model reaction and measured KIEs for the enzymic reaction suggests that the transition states are similar. 

Zheng, Y.-J. Bruice, T. C. A theoretical examination of the factors controlling the catalytic efficiency of a transmethylation enzyme Catechol 0-methyltransferase, J. Am. Chem. Soc. 1997,119, 8137-8145. 

Mechanistic aspects of the action of folate-requiring enzymes involve one-carbon unit transfer at the oxidation level of formaldehyde, formate and methyl (78ACR314, 8OMI2I6OO) and are exemplified in pyrimidine and purine biosynthesis. A more complex mechanism has to be suggested for the methyl transfer from 5-methyl-THF (322) to homocysteine, since this transmethylation reaction is cobalamine-dependent to form methionine in E. coli. 

Similarly, Crespi-Perellino et al. (13,15), using cell cultures of A. altissima and providing L-, D-, and D,L-[w(7Av/cz c- C lryptophan as the precursor, carried out tracer experiments and proved the biosynthetic pathway to canthin-6-one alkaloids to be as follows (Scheme 7) tryptophan )8-carboline-l -propionic acid — 4,5-dihydrocanlhin-6-one (29) canthin-6-one (1)—> l-hydroxycanthin-6-one (10) l-methoxycanthin-6-one (11) —> l-methoxycanthin-6-one 3-oxide (12). In the biosynthetic pathway to canthin-6-one alkaloids, oxidation proceeds stepwise. The hydroxyl group at position 1 of canthin-6-one is methylated, and 11 is readily formed this formation is considered to be a transmethylation promoted by a specific enzyme. 

Halogenated compounds are produced both by de novo synthesis involving direct incorporation of halide ion and by transmethylation reactions (Section 6.11.4). The former include reactions mediated by haloperoxidases in the presence of hydrogen peroxide and halide ion, and these enzyme systems have wide biosynthetic capability (Neidleman and Geigert 1986 van P6e 1996) including oxidative dimerization resulting, for example, in the formation of 2,3,7,8-tetrachlorodibenzo[l,4]dioxin from 2,4,5-trichlorophenol (Svenson et al. 1989). 

Vidarabine is an inhibitor of viral DNA synthesis. Cellular enzymes phosphorylate vidarabine to the triphosphate, which inhibits viral DNA polymerase activity in a manner that is competitive with deoxyadenosine triphosphate. Vidarabine triphosphate is incorporated into both cellular and viral DNA, where it may act as a chain terminator. Vidarabine triphosphate also inhibits ribonucleoside reductase, RNA polyadenylation, and 5 -adenosylhomocysteine hydrolase, an enzyme involved in transmethylation reactions. Resistant variants due to mutations in viral DNA polymerase can be selected in vitro. 

Since methionine has several pathways open to it, it is essential to know what factors control the direction that its metabolism takes. Studies in young adults have shown that the utilization of methyl groups is normally accounted for chiefly by creatinine formation. This reaction consumes more 5-adenosylmethionine than all other transmethylations together. However, examination of enzyme activities from these two pathways in fetal animals leads to the conclusion that remethylation preponderates over transsulfuration. Indeed, since y-cystathionase activity is immeasurable in human fetal liver and brain, not only is the remethylation sequence favored, but also cysteine then becomes an essential amino acid for the fetus and infant. 

Fig. 20.3 Pathway of methionine metabolism. The numbers represent the following enzymes or sequences (1) methionine adenosyltransferase (2) S-adenosylmethionine-dependent transmethylation reactions (3) glycine methyltransferase (4) S-adenosylhomocysteine hydrolase (5) betaine-homocysteine methyltransferase (6) 5-methyltetrahydrofolate homocysteine methyltransferase (7) serine hydroxymethyltransferase (8) 5,10-methylenetetrahydrofolate reductase (9) S-adenosylmethionine decarboxylase (10) spermidine and spermine synthases (11) methylthio-adenosine phosphorylase (12) conversion of methylthioribose to methionine (13) cystathionine P-synthase (14) cystathionine y-lyase (15) cysteine dioxygenase (16) cysteine suplhinate decarboxylase (17) hypotaurine NAD oxidoreductase (18) cysteine sulphintite a-oxoglutarate aminotransferase (19) sulfine oxidase. MeCbl = methylcobalamin PLP = pyridoxal phosphate...

The preceding paragraphs and figures to 2.11 present the highlights of tissue metabolism. The enzymes referred to in the text and on the figures are listed in the final section of this review together with others involved in transmethylation reactions, the transport of ions, and those involved in the synthesis and metabolism of chemical transmitter substances which will be discussed later. The enzymes of tissue metabolism listed in the final section are of direct interest to the biochemical toxicologist for a number of reasons. 

This enzyme catalyses the reaction that generates methionine from homocysteine, a metabolic step associated with transmethylation by iS-adenosylmethionine. 

This enzyme contains tightly bound cyanocobalamin which imparts a salmon pink colour. It catalyses one of the reactions which generate methionine from homocystein, and is thus involved indirectly in transmethylation. It may be assayed radiochemically. 

Later experimental work provided evidence that the 8-carbon polyketoacid intermediate in the synthesis of y-coniceine is derived from octanoic acid since this acid was shown to be readily incorporated into coniine. Further work indicated that 5-keto-octanoic acid and 5-keto-octanal were produced during the biosynthesis of y-coniceine. A transaminase (L-alanine 5-keto-octanal aminotransferase) was obtained from C. maculatum. This transaminase catalyzes the reaction between 5-keto-octanal with L-alanine as the amino group donor to form the piperidine ring and the propyl side chain. Another C. maculatum alkaloid, A-methylconiine, was shown to be produced by another enzyme from the plant a coniine methyltransferase which acts as a transmethylator utilizing 5-adenosyl-L-methionine as a methyl group donor. 

Nomicotine in N. glutinosa and in N. glauca is formed only in the leaves and at the expense of nicotine translocated from the roots as could be proved by reciprocal graft combinations with tomato (Solanum lycopersicum) shoots/roots (Dawson 1945). Already in this early report it was speculated that nicotine is converted to nor-nicotine probably by transmethylation . However, according to a proposal of Leete (1977), iV -formylnomicotine may be formed by oxidation of the iV-methyl group of nicotine followed by oxidation to nomicotine. Later a partial characterisation of nicotine iV-demethylase from microsomes of N. otophora was documented. Demethylation was interpreted to be associated with cytochrome P-450 rather than achieved by transmethylation (Bush et al. 1999 and references therein). The enzyme turned out to be NADPH-dependent in cell-free preparations from cell cultures of N. tabacum (Hao and Yeoman 1996b). Recently, it has been proved that CYP82E4 is involved in the metabolic conversion of nicotine to nomicotine in tobacco (Siminszky et al. 2005). 

The transmethylation reaction, in which methionine is of major importance, has been described in the chapter on Enzymes in Metabolic Sequences (see p. 55), to which the reader may refer. The outstanding reaction involving methionine and cysteine to be discussed in this section is transsulfuration. 

Fig. 10.1. Defects of transmethylation (methioninehomocysteine), transsulfuration (methionine sulfate), and remethylation (homocysteine - methionine) enzymes of sulfur amino acid metabolism 10.1, methionine adenosyltransferase 10.2, cystathionine ) -synthase 10.3, y-cystathionase 10.4, sulfite oxidase 10.5, molybdenum cofactor 10.6, methylenetetrahydrofolate reductase 10.7 and 10.8, methionine synthase.

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