Human pharmacogenetics of methyl conjugation

Methyl conjugation is an important pathway in drug metabolism. Activities of three human drug-metabolizing methyltransferase enzymes, catechol-O-methyltransferase (COMT) (EC 2.1.1.6), thiopurine methyltransferase ( TPMT ) (EC 2.1.1.67), and thiol methyltransferase (TMT) (EC 2.1.1.9), are controlled by inheritance. COMT activity in the red blood cell (RBC) is regulated by a single genetic locus with two alleles, COMTL for low activity and COMTH for high activity. Gene frequencies of these two alleles were approximately equal in a white population sample of Northern European origin. The genetically controlled level of COMT activity in the RBC reflects the level of enzyme activity in other tissues and is significantly correlated with individual variations in the methyl conjugation of catechol drugs such as L-dopa and methyldopa. TPMT catalyzes the S-methylation of thiopurines and thiopyrimidines . RBC TPMT activity is also controlled by a single genetic locus with two alleles, TPMTL for low and TPMTH for high activity. The gene frequencies of these two alleles were 0.06 and 0.94, respectively, in a white population sample. RBC TPMT activity reflects the level of enzyme activity in other cells and tissues such as the lymphocyte and kidney. TMT catalyzes the S-methylation of aliphatic sulfhydryl compounds such as the drugs captopril and D-penicillamine. The heritability of the level of RBC membrane TMT activity has been estimated on the basis of family studies to be approximately 0.98. Regulation of these three methyl-conjugating enzymes by inheritance raises the possibility that genetically determined methylator status may be one factor responsible for variations in drug metabolism in humans.     

Inheritance of low erythrocyte catechol-o-methyltransferase activity in man.

Catechol-O-methyltransferase activity was measured in blood obtained from 373 randomly selected subjects aged 16-18, 262 consecutive adult blood donors, and 201 first-degree relatives of subjects with RBC COMT activity of less than 8 U. The distribution of RBC COMT activity in a randoly selected populations was apparently bimodal with a nadir at approximately 8 U. Of a randomly selected population, 23% had low RBC COMT activity (less than 8 U), Because of previous reports of a significant sibling-sibling correlation of RBC COMT activity and because of the presence of a subgroup of subjects with low enzyme activity, RBC COMT activity was measured in blood from first-degree relatives of probands with low erythrocyte enzyme activity in 48 families. The results of segregation analyses of the data were compatible with autosomal recessive inheritence of an allele for low RBC COMT activity. RBC COMT in blood samples from siblings of probands inthese families also showed an apparent biomodal distribution.     

Developmental Patterns of Catechol-O-Methyltransferase in Genetically Different Rat Strains: Enzymatic and Immunochemical Studies

Abstract: Catechol- O -methyltransferase (COMT) activity in the liver and kidneys of adult Fischer-344 (F-344) rats is only half of that in the same organs of Wistar-Furth (W-F) rats. The trait of low COMT activity in these animals is inherited in an autosomal recessive fashion. A comprehensive study of patterns of change in COMT activity during growth and development was performed to determine whether "temporal gene" effects might play a role in the inherited differences in enzyme activity present in adult animals. The COMT activity expressed per mg protein in liver and kidneys of newborn F-344 rats is only 50–60% of that in the same organs of W-F animals. The liver and the kidneys of newborn rats of both strains have COMT activity an order of magnitude higher than those in brain, heart, or blood. In addition, in both strains there are much larger increases in liver and kidney COMT activities during growth and development (5–10 fold) than in blood, brain, or heart (one- to twofold). Immunotitration with antibodies against rat COMT demonstrates that differences in immunoreactive COMT parallel differences in COMT activity, both between strains and within strains during growth and development. However, when the temporal patterns of change in enzyme activities in the liver and the kidneys of the two strains of rat are compared at multiple times during growth and development, no differences in the patterns are present. These results make it unlikely that temporal gene effects can explain the inherited differences in COMT activity in liver and kidneys of F-344 and W-F rats.     

Catechol-O-methyltransferase: effects of the val108met polymorphism on protein turnover in human cells

A single nucleotide polymorphism in the human COMT (catechol-O-methyltransferase) gene has been associated with increased risk for breast cancer and several CNS diseases and disorders. The G to A polymorphism causes a valine (val) to methionine (met) substitution at codon 108 soluble - (S)/158 membrane - (MB)-COMT, generating alleles encoding high and low-activity forms of the enzyme, COMT H and COMT L, respectively. Tissues and cells with a COMT LL genotype have decreased COMT activity compared to COMT HH cells. Previously, we reported that the decreased activity was due to decreased amounts of S-COMT L protein in human hepatocytes. In this study, we investigated the role of S-COMT protein synthesis and turnover as determinates of reduced COMT protein in COMT LL compared to COMT HH cells. No association between S-COMT protein synthesis and COMT genotype was detected. Using a pulse-chase protocol, the half-life of S-COMT H was determined to be 4.7 days, which was considerably longer than expected from the half-lives of other phase 2 enzyme proteins. The half-life of S-COMT L compared to S-COMT H protein was significantly shorter at 3.0 days, but the difference was affected by the medium used during the chase period. These results suggest that increased turnover may contribute to reduced COMT activity in cells and tissues from COMT LL individuals. Subtle differences appear to be able to affect the stability of the S-COMT L protein, and this may contribute to the differences observed in epidemiological studies on the association of this polymorphism with breast cancer risk.     

Human erythrocyte catechol-O-methyltransferase: correlation with lung and kidney activity

Catechol-O-methyltransferase (COMT) activity measured in homogenates of human lung or renal cortical tissue was approximately an order of magnitude greater than the enzyme activity in human erythrocytes. Lung and blood enzyme activities varied from individual to individual over a 5–7 fold range. There was a highly significant correlation between the COMT activity in erythrocytes and the enzyme activity in lung (r = 0.59, P < .001, N = 29) and kidney (r = 0.81, P < .005, N = 12) of the same subjects. These results suggest that the relative COMT activity in lung and kidney might be predicted by the measurement of the enzyme activity in blood and raise the possibility that individual variation in the metabolism of endogenous catecholamines and catechol drugs in man may result in part from variations in COMT activity.     

Family studies of plasma dopamine-beta-hydroxylase thermal stability.

There are large individual variations in the thermal stability of human plasma dopamine-beta-hydroxylase (DBH). These variations are a characteristic of the DBH molecule itself. Individual subjects may be classified as those with thermolabile and those with thermostable plasma DBH. Of 362 randomly selected unrelated children, 8.01%, and of 238 randomly selected unrelated adult subjects, 5.46% had thermolabile plasma DBH. There was not a significant correlation of DBH thermolability with either sex or age on the basis of data from 230 adults and children in 53 randomly selected families. Subjects with thermolabile DBH had basal enzyme activity only about 55% of that in subjects with stable enzyme. There was not a direct relationship between DBH thermolability and the allele DBHL, the presence of which results in very low basal enzyme activity. There was a significant familial aggregation of the trait of DBH thermolability, but there was not a significant correlation of this trait among spouses. Although preliminary pedigree evaluation raised the possibility of monogenic inheritance of the trait of DBH thermolability by an autosomal recessive mechanism, three separate families in which both parents had thermolabile enzyme included offspring with thermostable DBH. All five of these offspring had very low basal plasma DBH and were presumed to be homozygous for the allele DBHL. These observations raised the possibility that the trait of plasma DBH thermolability may be inherited, and that there may be an interaction between the locus or loci responsible for thermal stability and the locus DBH.     

Functional and structural comparisons of cysteine residues in the Val108 wild type and Met108 variant of human soluble catechol O-methyltransferase

Catechol O-methyltransferase (COMT) plays an important role in the inactivation of biologically active and toxic catechols. This enzyme is genetically polymorphic with a wild type and a variant form. Numerous epidemiological studies have shown that the variant form is associated with an increased risk of developing estrogen-associated cancers and a wide spectrum of mental disorders. There are seven cysteine residues in human S-COMT, all of which exist as free thiols and are susceptible to electrophilic attack and/or oxidative damage leading to enzyme inactivation. Here, the seven cysteine residues were systematically replaced by alanine residues by means of site-directed mutagenesis. The native forms and cysteine/alanine mutants were assayed for enzymatic activity, thermal stability, methylation regioselectivity, and reactivity of cysteine residues to thiol reagent. Our data showed that although there is only one encoding base difference between these two COMT forms, this difference might induce structural changes in the local area surrounding some cysteine residues, which might further contribute to the different roles they might play in enzymatic activity, and to the different susceptibility to enzyme inactivation.     

Levels of uroporphyrinogen decarboxylase (URO-D) in erythrocytes of Italian porphyria cutanea tarda patients.

Porphyria cutanea tarda (PCT) is a human metabolic disorder due to the acquired or genetic impairment of uroporphyrinogen decarboxylase (URO-D) activity, the fifth enzyme of the heme biosynthetic pathway. A classification of inherited and non-inherited forms is based on the enzyme activity levels in red blood cells (RBC). Clinical manifestations of PCT are often precipitated by triggering factors such as alcohol, drug abuse, estrogens, virus infections, hepatotoxic chemicals and hepatic siderosis. We measured URO-D activity in RBC from a large sample of Italian PCT patients in order to define the enzyme activity distribution and to attempt a correlation among activity, risk factors and clinical outcome. Three classes of patients with low, normal and over-normal URO-D activity were defined according to control values. Low URO-D levels were present in 25.8% of patients, suggesting the familial form of PCT (type II). In this group, the outcome of PCT seems to be less influenced by risk factors. Patients with over-normal URO-D activity in RBC deserve further investigation.     

Characterization of wild-type and mutant forms of human tryptophan hydroxylase 2

Tryptophan hydroxylase (TPH) catalyses the rate-limiting step in the biosynthesis of serotonin. In vertebrates, the homologous genes tph1 and tph2 encode two different enzymes with distinct patterns of expression, enzyme kinetics and regulation. Variants of TPH2 have recently reported to be associated with reduced serotonin production and behavioural alterations in man and mice. We have produced the human forms of these enzymes in Esherichia coli and in human embryonic kidney cell lines (HEK293) and examined the effects of mutations on their heterologous expression levels, solubility, thermal stability, secondary structure, and catalytic properties. Pure human TPH2 P449R (corresponds to mouse P447R) had comparable catalytic activity (V(max)) and solubility relative to the wild type, but had decreased thermal stability; whereas human TPH2 R441H had decreased activity, solubility and stability. Thus, we consider the variations in kinetic values between wild-type and TPH2 mutants to be of secondary importance to their effects on protein stability and solubility. These findings provide potential molecular explanations for disorders related to the central serotonergic system, such as depression or suicidal behaviour.     

Catechol-O-methyltransferase biochemical genetics: Human lymphocyte enzyme

Human erythrocyte (RBC) catechol-O-methyltransferase (COMT) is under genetic control. Experiments were performed to determine whether COMT in the human lymphocyte is regulated in parallel with RBC COMT. Supernatants of lymphocyte homogenates contained COMT activity. However, they also contained a potent COMT inhibitor, the effect of which could be negated by dilution. Lymphocyte COMT activity was maximal at a reaction pH of 7.7 and at a MgCl₂ concentration of 0.67mm. The apparent K _m value for 3,4-dihydroxybenzoic acid, the catechol substrate for the reaction, was 1.2×10^?5 m and that for S-adenosyl-l-methionine, the methyl donor, was 2.3×10^?6 m. An average of 48.3±3.3% (mean ± SEM) of the enzyme activity in crude lymphocyte homogenates from 3 subjects was removed by centrifugation at 100,000 g for 1 hr and was presumed to be membrane associated. The average COMT activity in lymphocytes isolated from blood of 23 randomly selected adult subjects was 14.0±1.2 units/10⁶ cells (mean ± SEM) or 913±69 units/mg protein. There was a significant correlation of relative RBC with relative lymphocyte COMT activity in these 23 subjects. The correlation coefficient was 0.733 (P<0.001) when lymphocyte enzyme activity was expressed per milligram of protein and 0.649 (P<0.001) when lymphocyte activity was expressed per 10⁶ cells. These results are compatible with the conclusion that the genetic polymorphism which regulates RBC COMT activity may also regulate the level of human lymphocyte COMT activity.     

Phenol sulfotransferase inheritance

1. Phenol sulfotransferase (PST) catalyzes the sulfate conjugation of many phenolic and catechol neurotransmitters. Human tissues contain both thermostable (TS) and thermolabile (TL) forms of PST that differ in their substrate specificities, inhibitor sensitivities, physical properties, and regulation. 2. Individual variations in the levels of activity of both TS and TL PST in the human platelet are strongly influenced by inheritance. 3. Individual differences in the level of platelet TS PST activity are correlated with individual variations in the activity of this form of the enzyme in human cerebral cortex, liver, and intestinal mucosa. 4. There are also individual familial differences in the thermal stability of TS PST in the platelet. These differences are correlated with individual variations in the thermal stability of TS PST in cerebral cortex, liver, and intestinal mucosa. 5. Individual variations in the thermal stability of TS PST in hepatic tissue are associated with the presence of one or both of a pair of TS PST isozymes that can be separated by ion-exchange chromatography and that differ in their thermal stabilities. 6. This series of observations suggests that a structural gene polymorphism may be one mechanism by which inheritance controls TS PST in humans. The isozymes of TS PST in liver may represent the products of alternative alleles for this polymorphism, alleles that might control the structure of TS PST in many human tissues.     

Oxidative inhibition of human soluble catechol-O-methyltransferase

A common polymorphism in the human gene for catechol-O-methyltransferase results in replacement of Val-108 by Met in the soluble form of the protein (s-COMT) and has been linked to breast cancer and neuropsychiatric disorders. The 108M and 108V variants are reported to differ in their thermal stability, with 108M COMT losing catalytic activity more rapidly. Because human s-COMT contains seven cysteine residues and includes CXXC and CXXS motifs that are associated with thiol-disulfide redox reactions, we examined the effects of reducing and oxidizing conditions on the enzyme. In the absence of a reductant 108M s-COMT lost activity more rapidly than 108V, whereas in the presence of 4 mm dithiothreitol (DTT) we found no significant differences in the stability of the two variants at 37 degrees C. DTT also restored most of the activity that was lost upon incubation at 37 degrees C in the absence of DTT. Mass spectrometry showed that cysteines 188 and 191 formed an intramolecular disulfide bond when s-COMT was incubated with oxidized glutathione, whereas cysteines 69, 95, 157, and 173 formed protein-glutathione adducts. Replacing Cys-95 by serine protected 108M s-COMT against inactivation in the absence of a reductant; C33S and Cys-188 mutations had little effect, and C69S was destabilizing. The sequences surrounding the reactive cysteine residues of human s-COMT and other proteins that form glutathione adducts at identified sites all include Pro and/or Gly and most include a hydrogen-bonding residue, suggesting that glutathiolation at conserved sites plays a physiologically important role.     

Catechol-O-methyltransferase: Effects of the val108met polymorphism on protein turnover in human cells

A single nucleotide polymorphism in the human COMT (catechol-O-methyltransferase) gene has been associated with increased risk for breast cancer and several CNS diseases and disorders. The G to A polymorphism causes a valine (val) to methionine (met) substitution at codon 108 soluble - (S)/158 membrane - (MB)-COMT, generating alleles encoding high and low-activity forms of the enzyme, COMT^H and COMT^L, respectively. Tissues and cells with a COMT^LL genotype have decreased COMT activity compared to COMT^HH cells. Previously, we reported that the decreased activity was due to decreased amounts of S-COMT^L protein in human hepatocytes. In this study, we investigated the role of S-COMT protein synthesis and turnover as determinates of reduced COMT protein in COMT^LL compared to COMT^HH cells. No association between S-COMT protein synthesis and COMT genotype was detected. Using a pulse-chase protocol, the half-life of S-COMT^H was determined to be 4.7 days, which was considerably longer than expected from the half-lives of other phase 2 enzyme proteins. The half-life of S-COMT^L compared to S-COMT^H protein was significantly shorter at 3.0 days, but the difference was affected by the medium used during the chase period. These results suggest that increased turnover may contribute to reduced COMT activity in cells and tissues from COMT^LL individuals. Subtle differences appear to be able to affect the stability of the S-COMT^L protein, and this may contribute to the differences observed in epidemiological studies on the association of this polymorphism with breast cancer risk.     

THE REGIONAL AND SUBCELLULAR DISTRIBUTION OF CATECHOL-O-METHYL TRANSFERASE IN THE RAT BRAIN

Catechol-O-methyl transferase (COMT) activities determined in different regions of rat brain showed small variations. Highest activities were found in the hypothalamus and corpora quadrigemina, and lowest activities in the hippocampus and corpus striatum. The regional distribution of COMT was thus at variance with the distribution of DOPA decar- boxylase in this study and with the distribution of catecholamines and tyrosine hydroxylase reported in the literature. Determinations of the subcellular distribution of COMT in rat forebrain showed that 50 per cent of the activity was recovered in the high speed supernatant fluid and about 33 per cent in the crude mitochondrial fraction. Further separation of the latter by discontinuous sucrose gradients showed that the particulate COMT was found in the synaptosomal fraction in an occluded form. Full enzyme activity was only obtained after treatment with a detergent or after resuspension in water. After hypo-osmotic rupture of the crude mitochondrial fraction, COMT was recovered in the cytoplasmic fraction. The subcellular distribution of COMT was very similar to the ones of lactate dehydrogenase and DOPA decarboxylase. The proportions of soluble COMT obtained from homogenates of various regions of the brain differed from that of choline acetyl transferase and DOPA decarboxylase but were similar to that of lactate dehydrogenase. In conclusion, COMT is a cytoplasmic enzyme almost evenly distributed in the CNS. Its distribution does not resemble the distributions of the catecholamines or of the enzymes participating in the synthesis of catecholamines.     

Thermal denaturation of Micrococcus lysodeikticus adenosine triphosphatase. Influence of temperature on the circular dichroism, fluroescence and enzymic activity of the protein.

The soluble ATPase (adenosine triphosphatase) from Micrococcus lysodeikticus underwent a major unfolding transition when solutions of the enzyme at pH 7.5 were heated. The midpoint occurred at 46 degrees C when monitored by changes in enzymic activity and intrinsic fluorescence, and at 49 degrees C when monitored by circular dichroism. The products of thermal denaturation retained much secondary structure, and no evidence of subunit dissociation was detected after cooling at 20 degrees C. The thermal transition was irreversible, and thiol groups were not involved in the irreversibility. The presence of ATP, adenylyl imidodiphosphate, CaCl2 or higher concentrations of ATPase conferred stability against thermal denaturation, but did not prevent the irreversibility one denaturation had taken place. In the presence of guanidinium chloride, thermal denaturation occurred at lower temperatures. The midpoints of the transition were 45 degrees C in 0.25 M-, 38 degrees C in 0.5 M-and 30 degrees C in 0.75 M-denaturant. In the highest concentration of guanidinium chloride a similar unfolding transition induced by cooling was observed. Its midpoint was 9 degrees C, and the temperature of maximum stability of the protein was 20 degrees C. The discontinuities occurring the the Arrhenius plots of the activity of this enzyme had no counterpart in variations in the far-u.v. circular dichroism or intrinsic fluorescence of the protein at the same temperature.     

Enzyme immobilization using a cellulose-binding domain: properties of a beta-glucosidase fusion protein.

Using molecular genetic techniques, a fusion protein has been produced which contains the cellulose-binding domain (CBD) of an exoglucanase (Cex) from Cellulomonas fimi fused to a beta-glucosidase (Abg) from Agrobacterium sp. The CBD functions as an affinity tag for the simultaneous purification and immobilization of the enzyme on cellulose. Binding to cellulose was stable for prolonged periods at temperatures from 4 degrees C to at least 50 degrees C, at ionic strengths from 10 mM to greater than 1 M, and at pH values below 8. The fusion protein can be desorbed from cellulose with distilled water or at pH greater than 8. Immobilized enzyme columns of the fusion protein bound to cotton fibers exhibited stable beta-glucosidase activity for at least 10 days of continuous operation at temperatures up to 37 degrees C. At higher temperatures, the bound enzyme lost activity. The thermal stability of the fusion protein was greatly improved by immobilization. Immobilization did not alter the pH stability. Except for its ability to bind to cellulose, the properties of the fusion protein were virtually the same as those of the native enzyme.     

Characterization of a caffeic acid 3-O-methyltransferase from wheat and its function in lignin biosynthesis

Caffeic acid 3-O-methyltransferase (COMT) catalyzes the multi-step methylation reactions of hydroxylated monomeric lignin precursors, and is believed to occupy a pivotal position in the lignin biosynthetic pathway. A cDNA (TaCM) was identified from wheat and it was found to be expressed constitutively in stem, leaf and root tissues. The deduced amino acid sequence of TaCM showed a high degree of identity with COMT from other plants, particularly in SAM binding motif and the residues responsible for catalytic and substrate specificity. The predicted TaCM three-dimensional structure is very similar with a COMT from alfalfa (MsCOMT), and TaCM protein had high immunoreactive activity with MsCOMT antibody. Kinetic analysis indicated that the recombinant TaCM protein exhibited the highest catalyzing efficiency towards caffeoyl aldehyde and 5-hydroxyconiferaldehyde as substrates, suggesting a pathway leads to S lignin via aldehyde precursors. Authority of TaCM encoding a COMT was confirmed by the expression of antisense TaCM gene in transgenic tobacco which specifically down-regulated the COMT enzyme activity. Lignin analysis showed that the reduction in COMT activity resulted in a marginal decrease in lignin content but sharp reduction in the syringl lignin. Furthermore, the TaCM protein exhibited a strong activity towards ester precursors including caffeoyl-CoA and 5-hydroxyferuloyl-CoA. Our results demonstrate that TaCM is a typical COMT involved in lignin biosynthesis. It also supports the notion, in agreement with a structural analysis, that COMT has a broad substrate preference.     

Pharmacogenetics of methyl conjugation and thiopurine drug toxicity

Pharmacogenetics is the study of inherited variations in drug response Pharmacogenetics uses the techniques of pharmacology, population genetics, biochemical genetics and, most recently, molecular biology, to study the biological basis for individual variation in therapeutic response and in the occurrence of adverse reactions to medications. Most pharmacogenetic experiments deal with inherited differences in drug metabolism. The discussion here will review inherited variation in the activity of thiopurine methyltransferase, an enzyme that catalyzes the methyl conjugation of an important group of drugs, the thiopurines.