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BI 187004, an 11β-hydroxysteroid dehydrogenase 1 inhibitor, was administered once daily for 14 days to eight patients with type 2 diabetes mellitus. N-methylation was identified as a major biotransformation pathway. In four patients treated with BI 187004, the plasma exposure of an N-methylbenzimidazole metabolite [N-methylbenzimidazole regioisomer 1 (M1)] was 7-fold higher than the remaining four patients, indicating a substantial degree of metabolic variation. To identify the methyltransferase enzymes responsible for N-methylation, BI 187004 was incubated with human liver microsomes (HLM), human kidney microsomes (HKM), and their respective cytosolic preparations in the presence and absence of isoform-selective chemical inhibitors. Additionally, BI 187004 was incubated with several human recombinant methyltransferases: catechol O-methyltransferase (rhCOMT), histamine N-methyltransferase (rhHNMT), nicotinamide N-methyltransferase (rhNNMT), glycine N-methyltransferase (rhGNMT), and thiopurine S-methyltransferase (rhTPMT). M1 was principally observed in HLM and HKM incubations, minimally formed in liver and kidney cytosol, and not formed during incubations with recombinant methyltransferase enzymes. In all microsomal and cytosolic incubations, the formation of M1 was inhibited only by 2,3-dichloro-α-methylbenzylamine (DCMB), an inhibitor of thiol S-methyltransferase (TMT), providing evidence that TMT catalyzed the formation of M1. Interestingly, the N-methylbenzimidazole regioisomer (M14) was only observed in vitro, predominantly during incubations with human kidney cytosol and rhHNMT. The formation of M14 was inhibited by amodiaquine (an HNMT inhibitor) and DCMB, providing additional evidence that both HNMT and TMT catalyzed M14 formation. Overall, using BI 187004 as a substrate, this study demonstrates a novel TMT-mediated N-methylation biotransformation and an HNMT-mediated regioselective N-methylation.

In humans, approximately two-thirds of the metabolic clearance of ziprasidone proceeds through reduction of the N-S bond of the benzisothiazole moiety, which is followed by methylation of the resulting thiophenol to the major metabolite S-methyldihydroziprasidone. The objective of this study was to gain an understanding of the underlying mechanism of this clearance route. Incubation of ziprasidone in human liver cytosol yielded the reduction product dihydroziprasidone. Heating the cytosol prior to incubation prevented the reaction, and the reaction was saturable (K(M) = 3.9 µM; V(max) = 0.24 nmol/min/mg protein), supporting that it was enzyme catalyzed. It was partially stimulated by 2-hydroxypyrimidine and inhibited by menadione, supporting a role for aldehyde oxidase in this reaction. However, incubation of ziprasidone with reduced glutathione, even in the absence of cytosol, readily yielded dihydroziprasidone indicating that there is also a chemical reduction component to this clearance pathway. The pathway was reversible because incubation of dihydroziprasidone in cytosol or with oxidized glutathione in buffer yielded ziprasidone. The methylation of dihydroziprasidone was observed in human liver microsomes in the presence of S-adenosylmethionine (K(M) = 14 µM; V(max) = 0.032 nmol/min/mg protein). This reaction was inhibited by 2,3-dichloro-α-methylbenzylamine supporting that thiol methyltransferase is the enzyme responsible for this reaction. Thus, the main metabolic pathway for ziprasidone in humans occurs via chemical reduction and aldehyde oxidase catalyzed reduction, followed by thiol methyltransferase catalyzed methylation.

Several marine algae including diatoms exhibit S-adenosyl-l-methionine (SAM) halide/thiol methyltransferase (HTMT) activity, which is involved in the emission of methyl halides. In this study, the in vivo biogenic emission of methyl iodide from the diatom Phaeodactylum tricornutum was found to be clearly correlated with iodide concentration in the incubation media. The gene encoding HTMT (Pthtmt) was isolated from P. tricornutum CCAP 1055/1, and expressed in Escherichia coli. The molecular weight of the enzyme was 29.7kDa including a histidine tag, and the optimal pH was around pH 7.0. The kinetic properties of recombinant PtHTMT towards Cl(-), Br(-), I(-), [SH](-), [SCN](-), and SAM were 637.88mM, 72.83mM, 8.60mM, 9.92mM, 7.9mM, and 0.016mM, respectively, and were similar to those of higher-plant HTMTs, except that the activity towards thiocyanate was lower. The biogenic emission of methyl halides from the cultured cells and the enzymatic properties of HTMT suggest that the HMT/HTMT reaction is key to understanding the biogenesis of methyl halides in oceanic environments as well as terrestrial ones.

BACKGROUND—Luminal anionic sulphide may contribute to epithelial damage in ulcerative colitis. Thiol methyltransferase (TMT) governs sulphide detoxification by the colonic mucosa and circulating erythrocytes. AIMS—To measure levels of TMT activity in erythrocytes of surgically treated cases of colitis or in rectal biopsies of defined groups of colitis. PATIENTS—Venepuncture blood was obtained from 37 blood donors and 27 subjects who had previously undergone a proctocolectomy for colitis: 18 for ulcerative colitis and nine for Crohn's colitis. Rectal biopsies from 122 cases were obtained: 47 without mucosal disease, 33 post-colon resection for cancer, 14 with moderate to severe ulcerative colitis, 15 with quiescent ulcerative colitis, seven with acute Crohn's colitis, and six with radiation proctitis. METHODS—TMT activity was measured by high performance liquid chromatography with radioactive detection to measure 14C methylmercaptoethanol formation, the reaction product of cell extracts incubated with mercaptoethanol and 14C S-adenosylmethionine. RESULTS—Erythrocyte TMT activity of surgically treated cases of colitis was significantly elevated (p

The physiological relevance of a novel thiol methyltransferase from cabbage, and its possible role in sulphur metabolism have been investigated. The enzyme was absent from the chloroplast, the site of sulphate reduction, and was localized in the cytosol. Potential substrates were initially screened on the basis of their ability to inhibit the methylation of iodide, a previously known substrate for the enzyme. Thiocyanate, 4,4′-thiobisbenzenethiol, thiophenol, and thiosalicylic acid were identified as possible substrates. Methylation of these thiols by the purified enzyme using [Methyl-3H]S-adenosyl- L-methionine confirmed their nature as substrates. The purified enzyme strongly preferred thiocyanate as a methyl acceptor. The enzyme had Km values of 11, 51, 250 and 746 mmol m−3 for thiocyanate, 4,4′-thiobisbenzenethiol, thiophenol and thiosalicylic acid, respectively. The identity of methylthiocyanate as the product of thiocyanate methylation by the purified enzyme was confirmed by mass spectrometry. The enzyme was strictly associated with glucosinolate-containing plants. Thiol substrates of the enzyme are known products of glucosinolate hydrolysis. Our observations indicate that this enzyme could be involved in the detoxification of reactive thiols produced upon glucosinolate degradation in these plants.

Two enzymes of detoxification were studied in blood samples from 27 patients with ulcerative colitis (UC) and 18 controls to determine whether there is an abnormality in sulfur metabolism in UC. Thiol methyltransferase (TMT) activity was measured in erythrocyte membranes as the extent of conversion of 2-mercaptoethanol to S-methyl-2-mercaptoethanol with [3H]methyl-S-adenosyl methionine as methyl donor. Phenol sulfotransferase (PST) activity was measured in platelet homogenates as the extent of sulfation of p-nitrophenol with 3-phosphoadenosine 5-phospho[35S]sulfate (PAPS) as sulfate donor. TMT activity was significantly higher in UC patients (27.0 vs 17.1 nmol/mg protein/hr; P < 0.005). No difference in PST activity was found. We conclude that TMT may be up-regulated in UC to detoxify excess hydrogen sulfide exposed to the peripheral blood compartment. This may arise from either increased luminal sulfide production or reduced colonic detoxification.

AIMS--To verify the improved thiol methyltransferase (TMT) assay and measure activity in isolated colonocytes and erythrocyte membranes of the same subjects. METHODS--High performance liquid chromatography with radioactivity detection was used to measure 14C-methylmercaptoethanol formation, the reaction product of cell extracts incubated with mercaptoethanol and 14C-S-adenosylmethionine. RESULTS--Verification of radiolabelled 14C-methylmercaptoethanol was by exogenous addition of methylmercaptoethanol and simultaneous ultraviolet detection at 214 nm. Using a substrate concentration of 10 mM mercaptoethanol, the Km for S-adenosylmethionine was 25 microM. The sensitivity of the radioactive method was 2 pmol, with coefficients of variation of 7% within assay and 6.4% between assay. TMT activities (mean +/- SE; n = 17) were 471 +/- 64 pmol/hour/mg protein for colonocytes and 73 +/- 7 pmol/hour/mg protein for erythrocyte membranes. CONCLUSIONS--The direct assay of TMT activity is sensitive, specific and eliminates concern over non-enzymatic methylation of thiol compounds. High activities in colonic epithelial cells deserve evaluation in disease states.

Cysteine conjugate β-lyase activity was demonstrated in the cuticle of M. sexta and H. virescens and was present in the cytosolic fraction from both of these species. Enzymatic activity was significantly decreased following each molt of the larvae and the increased until the start of the next instar. β-Lyase activity increased for 20 h after eclosure in the adult and the highest larval activity was measured in the late fifth instar. The insect enzyme appears to be less sensitive to (aminooxy)acetic acid, a cysteine conjugate β-lyase inhibitor, than was reported for the rat renal enzyme. Thiol S-methyltransferase activity was also localized in the cuticle of both insect species but was present in both the microsomal and cytosolic fractions.

Ulcerative colitis is associated with a selective reduction of n-butyrate oxidation by the colonic epithelial cells although the reason for this has been unclear. Colonic epithelial cell n-butyrate oxidation can be inhibited in vitro by incubation with sulphide but the role of mucosal detoxification of sulphide in the metabolic welfare of the colonic mucosa has not been examined. This study aimed to assess the role mucosal detoxification of sulphide by thiolmethyltransferase (TMT)-mediated methylation may play in protecting the healthy colonic mucosa from the adverse effects of luminal sulphide. Colonic epithelial cell suspensions from healthy human proximal (n=9) and distal colon (n = 10) were incubated in the presence of 14 C-labelled n-butyrate (5 mmol/L) alone, butyrate plus sodium hydrogen sulphide (NaHS) (1.5 mmol/L), or butyrate plus NaHS plus S-adenosyl-methionine 1,4 butane disulphonate (SAMe) (5 mmol/L). Study end points were metabolic performance ( 14 CO 2 production) and mucosal TMT activity. Incubation with NaHS induced a significant inhibition of 14 CO 2 production compared with control incubations (P< 0.001) which was similar for proximal and distal colonic cell suspensions. S-adenosyl-methionine 1,4 butane disulphonate reversed this effect completely in proximal but not in distal cell incubations, suggesting a greater susceptibility of the distal colon to the sulphide effect. Although median whole mucosal TMT values did not differ between proximal and distal colonic mucosa, a non-normal distribution of distal TMT values was observed. However, neither the degree of sulphide inhibition of control 14 CO 2 production nor the degree to which SAMe reversed this inhibition correlated with whole mucosal TMT activity. The study concluded that regional variation exists in TMT activity in the human colon but whilst methylation appears to protect colonic epithelial cells against sulphide-induced inhibition of n-butyrate oxidation, this cannot be directly correlated with mucosal TMT activity.

1. Human hepatic thiol methyltransferase (TMT) is a microsomal enzyme known to catalyse the in vitro S-methylation of diethyldithiocarbamic acid (DDC) to form diethyldithiocarbamic acid methyl ester (methyl DDC). In vivo data are needed to investigate further the biotransformation of DDC to methyl DDC. Thus, we have characterized the in vitro conversion of DDC to methyl DDC using rat liver microsomes with the ultimate goal of establishing an animal model. 2. Formation of methyl DDC in rat liver microsomes was confirmed by hplc analysis. 3. Rat liver microsomes catalysed methylation of DDC with low and high Km's of 5 +/- 6 and 260 +/- 80 microM respectively and with corresponding Vmax's of 0.09 +/- 0.05 and 0.59 +/- 0.04 nmol/min/mg protein. 4. Rat liver TMT activity was maximally inhibited by 57 +/- 6% by 1000 microM 2,3-dichloro-alpha-methylbenzylamine (DCMB), whereas human TMT was completely inhibited. The concentration of half maximal inhibition of rat TMT for DCMB was 2 microM. 5. Incomplete inhibition of rat TMT activity by DCMB suggests a possible alternative pathway of methylation.

There are reports that patients with Parkinson9s disease (PD) and Alzheimer9s disease (AD) have reduced levels of thiolmethyltransferase (TMT) in erythrocyte membranes.TMT methylates thiols and thiocarbamates, thereby reducing their toxicity. We examined TMT levels in erythrocytes from patients with PD and AD and from age-matched controls. Specific activities of TMT were 564 plus minus 199 U/mg protein in PD (n equals 32), 513 plus minus 118 in AD (n equals 13), and 565 plus minus 183 in controls (n equals 35). There was no difference between any of the groups (p equals 0.64). We failed to confirm TMT as a marker for neurodegenerative diseases or for this metabolic defect predisposing to susceptibility to neurotoxins. NEUROLOGY 1995;45: 1903-1906

Thioether methyltransferase (S-adenosyl-l-methionine: thioether S-methyltransferase; EC 2.1.1.96) catalyzes the methylation of X in compound of the type R-X-R′(X = S, Se, Te), yielding a methyl onium ion. Previous results using mice have demonstrated a role for thioether methyltransferase in the conversion and clearance of thioethers by methylation to more water-soluble methyl sulfonium ions suitable for excretion in the urine. A potential major physiological source of thioethers is reactions catalyzed by microsomal thiol methyltransferase (S-adenosyl-l-methionine: thiol S-methyltransferase; EC 2.1.1.9), which has been shown to methylate a diverse range of aliphatic sulfhydryl compounds. This study provides evidence for the sequential methylation of the aliphatic thiol, 2-mercaptoethanol, first to the methyl thioether, 2-(methylthio)ethanol, by thiol methyltransferase followed by methylation of this methyl thioether to the dimethyl sulfonium ion, 2-(dimethylthio)ethanol, by thioether methyltransferase. This sequence of reactions was demonstrated in vivo by injecting mice i.p. with radioactive 2-mercaptoethanol and analyzing the labeled methylated products, 2-(methylthio)ethanol and 2(dimethylthio)ethanol, in the urine by HPLC. In addition, the system converting 2-mercaptoethanol to 2-(dimethylthio)ethanol was reconstituted in vitro using solubilized mouse liver microsomes as a source of thiol methyltransferase and purified thioether methyltransferase from mouse lung. The results of these in vivo and in vitro studies established the sequential methylation of 2-mercaptoethanol by these two enzymes.

A new transmethylating enzyme system in mammalian tissues, catalyzing methyl transfer from S-adenosylmethionine to sulfhydryl compounds, has been studied. A series of “nonphysiological” sulfhydryl compounds (BAL, mercaptoethanol, O-methyl-mercaptoethanol, mercaptoacetic acid, β-mercaptoproprionic acid, methymercaptan and hydrogen sulfide) have been found to act as methyl accepting substrates, whereas “physiological” sulfhydryl compounds (homocysteine, cysteine and glutathione) are inactive. With S-adenosylethionine a “transethylation” is found to take place. The transmethylating enzyme system is present in the microsome fraction of several tissues of the rat and in the liver of 6 mammalian species investigated.

Microsomal thiol S-methyltransferase (TMT) of rat brain catalysed the methylation of dithiothreitol (Km = 84 microM) and other thiol compounds using S-adenosylmethionine as methyl donor (Km = 3.7 microM). With increasing polarity of thiol substrates there was a decrease in the maximal velocities of reaction and an increase in the apparent Km values. TMT was found to be unevenly distributed amongst various brain regions, with highest activities in the medulla oblongata and the hippocampus.

The thiol S-methyltransferase from rat liver has been solubilized and prepared in homogeneous form. The enzyme exists in a monomer of Mr 28,000 although enzyme activity is highly unstable with a half-life of 4 days under the best conditions of storage. The reaction requires S-adenosylmethionine as methyl donor but, as is the case with many enzymes active in detoxification, a large variety of lipophilic compounds can serve as acceptors. Acceptor activity is limited to thiols. The naturally occurring hydrophilic thiols, glutathione and cysteine, act neither as substrates nor as inhibitors. The course of the reaction is biphasic with an initial rapid formation of product that is followed by a slower linear rate. The suggestion is offered that this behavior reflects the slow dissociation of an enzyme-product complex composed of enzyme and S-adenosyl-homocysteine.

The enzymatic methylation of foreign sulphhydryl compounds in the microsomal fraction of mammalian liver was first reported by Bremer and Greenberg [2]. A broad degree of acceptor specificity was established, although "physiological" sulphhydryl-containing substances such as cysteine were inactive. On the other hand, structural requirements for the methyl donor molecule, S-adenosyl methionine, have been found to be quite specific [1] such that even close analogues are inactive, or at best inhibitory. A preliminary report from our laboratory has established the methylating capability of mammalian liver microsomes towards alkane thiols [4]. In contrast to the acceptors examined in earlier work, low members of the alkane thiol series are of endogenous physiological importance [3]. Methylation, which leads to blocking of the reactive sulphhydryl group can be considered as a detoxification process, and the activities observed in three independent studies on microsomes probably derive from a single enzyme or group of enzymes categorised by E.C. 2.1.1.9, S-adenosyl-L-methionine : thiol-S-methyltransferase. Straight-chain alkane thiols (C1-C10) and branchedchain isomers (C3-C~), and S-adenosyl methionine were purchased from commercial sources. The methyl thioether derivatives, required as reference samples, were synthesised from the respective alkyl bromides in reaction with sodium methyl thiolate. The microsomal fraction of mammalian liver homogenates was prepared by standard differential centrifugation techniques. The novelty of the present study lies partly in the method of assay of the methylation reaction, which in contrast to previous methods [1,2] does not require radioactivelylabelled substances. Incubations were carried out in sealed vials of a total volume 7 ml. The aqueous phase (1 ml) contained 1 mg of microsomal protein and 1 lamole of S-adenosyl methionine in 100 retool/1 of tris/HC1 buffer, pH 8. The system was thermostatted at 25 ~ C, and incubation was initiated by injection of the required amount of thin through the septum of the vial. Analysis was performed under the conditions reported previously [4] with removal of 5 gl of the gas phase in a gas-tight syringe and direct injection into the gas-chromatograph. Decrease in substrate and increase in product as a function of time could be monitored simultaneously as shown for ethane thiol in Fig. I, and integrated peak areas were converted into absolute quantities from calibration curves. The inset in the figure shows the kinetics ofmethylation for four different initial amounts of ethane thiol (So), whereby the gas-chromatograms in the figure represent one run. The degree of product formation, expressed as the amount of product at a given time, Pt, divided by the sum of product and residual substrate, (P, + S0, is plotted as a function of time, t. After an initial induction period, the plots were linear for at least 2 h. The quantity Pt/(l~t-Jr-St) is used rather than Pt directly to nullify errors arising from varying injected volumes from sample to sample. The slope of the plot, m, is thus equal to Pt/(Pt + St)" t. In the early stages of the reaction, Pt ~ St, so that 5', = So. Thus