The histamine N-methyltransferase T105I polymorphism affects active site structure and dynamics

Histamine N-methyltransferase (HNMT) is the primary enzyme responsible for inactivating histamine in the mammalian brain. The human HNMT gene contains a common threonine-isoleucine polymorphism at residue 105, distal from the active site. The 105I variant has decreased activity and lower protein levels than the 105T protein. Crystal structures of both variants have been determined but reveal little regarding how the T105I polymorphism affects activity. We performed molecular dynamics simulations for both 105T and 105I at 37 degrees C to explore the structural and dynamic consequences of the polymorphism. The simulations indicate that replacing Thr with the larger Ile residue leads to greater burial of residue 105 and heightened intramolecular interactions between residue 105 and residues within helix alpha3 and strand beta3. This altered, tighter packing is translated to the active site, resulting in the reorientation of several cosubstrate-binding residues. The simulations also show that the hydrophobic histamine-binding domain in both proteins undergoes a large-scale breathing motion that exposes key catalytic residues and lowers the hydrophobicity of the substrate-binding site.     

Theoretical 3D model of histamine N-methyltransferase: insights into the effects of a genetic polymorphism on enzymatic activity and thermal stability

Histamine N-methyltransferase (HNMT) catalyzes the N-methylation of histamine in mammals. The experimentally determined HNMT three-dimensional (3D) structure is not available. However, there is a common genetic polymorphism for human HNMT (Thr105Ile) that reduces enzymatic activity and is a risk factor for asthma. To obtain insights into mechanisms responsible for the effects of that polymorphism on enzymatic activity and thermal stability, we predicted the 3D structure of HNMT using the threading method and molecular dynamics simulations in water. Herein, we report a theoretical 3D model of human HNMT which reveals that polymorphic residue Thr105Ile is located in the turn between a beta strand and an alpha helix on the protein surface away from the active site of HNMT. Ile105 energetically destabilizes folded HNMT because of its low Chou-Fasman score for forming a turn conformation and the exposure of its hydrophobic side chain to aqueous solution. It thus promotes the formation of misfolded proteins that are prone to the clearance by proteasomes. This information explains, for the first time, how genetic polymorphisms can cause enhanced protein degradation and why the thermal stability of allozyme Ile105 is lower than that of Thr105. It also supports the hypothesis that the experimental observation of a significantly lower level of HNMT enzymatic activity for allozyme Ile105 than that with Thr105 is due to a decreased concentration of allozyme Ile105, but not an alternation of the active-site topology of HNMT caused by the difference at residue 105.     

Polymorphisms of two histamine-metabolizing enzymes genes and childhood allergic asthma: a case control study

Background

Histamine-metabolizing enzymes (N-methyltransferase and amiloride binding protein 1) are responsible for histamine degradation, a biogenic amine involved in allergic inflammation. Genetic variants of HNMT and ABP1 genes were found to be associated with altered enzyme activity. We hypothesized that alleles leading to decreased enzyme activity and, therefore, decreased inactivation of histamine may be responsible for altered susceptibility to asthma.

Methods

The aim of this study was to analyze polymorphisms within the HNMT and ABP1 genes in the group of 149 asthmatic children and in the group of 156 healthy children. The genetic analysis involved four polymorphisms of the HNMT gene: rs2071048 (-1637T/C), rs11569723 (-411C/T), rs1801105 (Thr105Ile = 314C/T) and rs1050891 (1097A/T) and rs1049793 (His645Asp) polymorphism for ABP1 gene. Genotyping was performed with use of PCR-RFLP. Statistical analysis was performed using Statistica software; linkage disequilibrium analysis was done with use of Haploview software.

Results

We found an association of TT genotype and T allele of Thr105Ile polymorphism of HNMT gene with asthma. For other polymorphisms for HNMT and ABP1 genes, we have not observed relationship with asthma although the statistical power for some SNPs might not have been sufficient to detect an association. In linkage disequilibrium analysis, moderate linkage was found between -1637C/T and -411C/T polymorphisms of HNMT gene. However, no significant differences in haplotype frequencies were found between the group of the patients and the control group.

Conclusions

Our results indicate modifying influence of histamine N-methyltransferase functional polymorphism on the risk of asthma. The other HNMT polymorphisms and ABP1 functional polymorphism seem unlikely to affect the risk of asthma.     

Association of Histamine N-Methyltransferase Thr105Ile Polymorphism with Parkinson’s Disease and Schizophrenia in Han Chinese: A Case-Control Study

Parkinson’s disease (PD) and schizophrenia (SCZ) are frequent central nervous disorders that have unclear etiologies but that show similarities in their pathogenesis. Since elevated histamine levels in the brain have been associated with PD and SCZ, we wanted to explore whether the Thr105Ile substitution in the histamine N-methyltransferase gene (HNMT-Thr105Ile), which impairs histamine degradation, is associated with either disease. We used the ligase detection reaction to genotype a case-control cohort of Han Chinese patients with PD or SCZ and healthy controls at the HNMT-Thr105Ile locus. The Ile allele was associated with reduced risk of PD (OR 0.516, 95%CI 0.318 to 0.838, p = 0.007) and of SCZ (OR 0.499, 95%CI 0.288 to 0.865, p = 0.011). Genotype frequencies and minor allele frequencies were similar between patients and controls when we compared males with females or early-onset patients with late-onset ones. Genotype and allele frequencies were not significantly different between PD patients with dyskinesia and PD patients without dyskinesia. Our results suggest that the heterozygous Thr/Ile genotype at the HNMT-Thr105Ile locus and the minor Ile105 allele protect against PD and SCZ in Han Chinese.     

Mechanisms for auto-inhibition and forced product release in glycine N-methyltransferase: crystal structures of wild-type, mutant R175K and S-adenosylhomocysteine-bound R175K enzymes

Glycine N-methyltransferase (S-adenosyl-l-methionine: glycine methyltransferase, EC 2.1.1.20; GNMT) catalyzes the AdoMet-dependent methylation of glycine to form sarcosine (N-methylglycine). Unlike most methyltransferases, GNMT is a tetrameric protein showing a positive cooperativity in AdoMet binding and weak inhibition by S-adenosylhomocysteine (AdoHcy). The first crystal structure of GNMT complexed with AdoMet showed a unique "closed" molecular basket structure, in which the N-terminal section penetrates and corks the entrance of the adjacent subunit. Thus, the apparent entrance or exit of the active site is not recognizable in the subunit structure, suggesting that the enzyme must possess a second, enzymatically active, "open" structural conformation. A new crystalline form of the R175K enzyme has been grown in the presence of an excess of AdoHcy, and its crystal structure has been determined at 3.0 A resolution. In this structure, the N-terminal domain (40 amino acid residues) of each subunit has moved out of the active site of the adjacent subunit, and the entrances of the active sites are now opened widely. An AdoHcy molecule has entered the site occupied in the "closed" structure by Glu15 and Gly16 of the N-terminal domain of the adjacent subunit. An AdoHcy binds to the consensus AdoMet binding site observed in the other methyltransferase. This AdoHcy binding site supports the glycine binding site (Arg175) deduced from a chemical modification study and site-directed mutagenesis (R175K). The crystal structures of WT and R175K enzymes were also determined at 2.5 A resolution. These enzyme structures have a closed molecular basket structure and are isomorphous to the previously determined AdoMet-GNMT structure. By comparing the open structure to the closed structure, mechanisms for auto-inhibition and for the forced release of the product AdoHcy have been revealed in the GNMT structure. The N-terminal section of the adjacent subunit occupies the AdoMet binding site and thus inhibits the methyltransfer reaction, whereas the same N-terminal section forces the departure of the potentially potent inhibitor AdoHcy from the active site and thus facilitates the methyltransfer reaction. Consequently GNMT is less active at a low level of AdoMet concentration, and is only weakly inhibited by AdoHcy. These properties of GNMT are particularly suited for regulation of the cellular AdoMet/AdoHcy ratio.     

Crystal structure of human L-isoaspartyl-O-methyl-transferase with S-adenosyl homocysteine at 1.6-A resolution and modeling of an isoaspartyl-containing peptide at the active site

Spontaneous formation of isoaspartyl residues (isoAsp) disrupts the structure and function of many normal proteins. Protein isoaspartyl methyltransferase (PIMT) reverts many isoAsp residues to aspartate as a protein repair process. We have determined the crystal structure of human protein isoaspartyl methyltransferase (HPIMT) complexed with adenosyl homocysteine (AdoHcy) to 1.6-A resolution. The core structure has a nucleotide binding domain motif, which is structurally homologous with the N-terminal domain of the bacterial Thermotoga maritima PIMT. Highly conserved residues in PIMTs among different phyla are placed at positions critical to AdoHcy binding and orienting the isoAsp residue substrate for methylation. The AdoHcy is completely enclosed within the HPIMT and a conformational change must occur to allow exchange with adenosyl methionine (AdoMet). An ordered sequential enzyme mechanism is supported because C-terminal residues involved with AdoHcy binding also form the isoAsp peptide binding site, and a change of conformation to allow AdoHcy to escape would preclude peptide binding. Modeling experiments indicated isoAsp groups observed in some known protein crystal structures could bind to the HPIMT active site.     

Membrane-bound histamine N-methyltransferase in mouse brain: possible role in the synaptic inactivation of neuronal histamine

In the CNS, histamine is a neurotransmitter that is inactivated by histamine N-methyltransferase (HNMT), a soluble enzyme localized to the cytosol of neurons and endothelial cells. However, it has not been established how extracellular histamine, a charged molecule at physiological pH, reaches intracellular HNMT. Present studies investigated two potential routes of histamine inactivation in mouse brain nerve terminal fractions (synaptosomes): (i) histamine uptake and (ii) histamine metabolism by HNMT. Intact synaptosomes demonstrated a weak temperature-dependent histamine uptake (0.098 pmol/min-mg protein), but contained a much greater capacity to metabolize histamine by HNMT (1.4 pmol/min-mg protein). Determination of the distribution of HNMT within synaptosomes revealed that synaptosomal membranes (devoid of soluble HNMT) contribute HNMT activity equivalent to intact synaptosomes (14.3 +/- 2.2 and 18.2 +/- 4.3 pmol/min-tube, respectively) and suggested that histamine-methylating activity is associated with the membrane fraction. Additional experimental findings that support this hypothesis include: (i) the histamine metabolite tele-methylhistamine (tMH) was found exclusively in the supernatant fraction following an HNMT assay with intact synaptosomes; (ii) the membrane-bound HNMT activity was shown to increase 6.5-fold upon the solubilization of the membranes with 0.1% Triton X-100; and (iii) HNMT activity from the S2 fraction, ruptured synaptosomes, and synaptosomal membranes displayed different stability profiles when stored over 23 days at - 20 degrees C. Taken together, these studies demonstrate functional evidence for the existence of membrane-bound HNMT. Although molecular studies have not yet identified the nature of this activity, the present work suggests that levels of biologically active histamine may be controlled by an extracellular process.     

Quantitative evaluation and regulation of S-adenosylmethionine-dependent transmethylation in sheep tissues

The overall rates of S-adenosylmethionine (AdoMet)-dependent transmethylation were estimated in various tissues from the initial rate of S-adenosylhomocysteine (AdoHcy) plus AdoMet accumulation after blocking hydrolysis of AdoHcy. The rates were found to differ widely among the tissues of sheep and the highest rate was in the pancreas, being 600 times higher than that in the muscle. Sheep liver possessed approximately 75% of total-body capacity for transmethylation although the transmethylation rate was approximately half that in rat liver. The minimum estimate of daily requirement of AdoMet for transmethylation for adult sheep was approximately 18 mmol, far in excess of methionine intake. Methionine loading elevated AdoMet levels only in the tissues with a high or moderate rate of transmethylation. The kinetic properties of major methyltransferases in sheep liver along with tissue distribution of AdoMet and AdoHcy suggest that transmethylation rate is subject to physiological regulation by tissue levels of AdoMet and AdoHcy.     

Kinetic mechanism of isoprenylated protein methyltransferase.

The kinetic mechanism of the rod outer segment (ROS) isoprenylated protein methyltransferase was investigated. This S-adenosyl-L-methionine (AdoMet)-linked enzyme transfers methyl groups to carboxyl-terminal isoprenylated cysteine residues of proteins, generating methyl esters. The enzyme also processes simple substrates such as N-acetyl-S-farnesyl-L-cysteine (L-AFC). Initial studies showed that a ping-pong Bi Bi mechanism could be eliminated. In a ping-pong Bi Bi mechanism plots of 1/v versus 1/[substrate A] at different fixed substrate B concentrations are expected to yield a family of parallel lines whose slopes equal Km/Vmax. In fact, converging curves were found, which suggested a sequential mechanism. Dead-end inhibitors were used in order to further investigate the kinetic mechanism. S-Farnesylthioacetic acid is shown to be a dead-end competitive inhibitor with respect to the prenylated substrate L-AFC. On the other hand, S-farnesylthioacetic acid proved to be uncompetitive with respect to AdoMet, suggesting an ordered mechanism with AdoMet binding first. Further evidence for this mechanism came from product inhibition studies using the methyl ester of L-AFC (L-AFCMe) and S-adenosyl-L-homocysteine (AdoHcy). Since AdoMet binds first to the enzyme, one of the products (L-AFCMe or AdoHcy) should be a competitive inhibitor with respect to it. It could be shown that AdoHcy is a competitive inhibitor with respect to AdoMet, but L-AFCMe is a mixed-type inhibitor both with respect to AdoMet and to L-AFC. Therefore, AdoHcy combines with the same enzyme form as does AdoMet, and must be released from the enzyme last. Moreover, L-AFC and L-AFCMe must bind to different forms of the enzyme.     

S-adenosylmethionine, S-adenosylhomocysteine and S-adenosylhomocysteine hydrolase variations during differentiation of Dictyostelium discoideum

We have analyzed the level of substrate (AdoMet) and products (AdoHcy) of transmethylations throughout the developmental cycle of the primitive eukaryote Dictyostelium discoideum. The ratio AdoMet/AdoHcy varied dramatically during differentiation. The intracellular level of AdoHcy decreased sharply after the beginning of starvation reaching a value of 18% of that in vegative cells within 4 h. In contrast, there was a two-fold transient increase in AdoMet at the time of aggregation. However, these changes were not related to changes in AdoHcy hydrolase since constant levels of both the protein and the activity were found until 16 h of differentiation. In particular, there was no indication of an in vivo inactivation of the enzyme by cAMP at the time of aggregation. These results are discussed with respect to the previously postulated role of AdoHcy hydrolase in the regulation of the AdoMet/AdoHcy ratio in eukaryotic cells.     

Kinetic and catalytic mechanism of HhaI methyltransferase

Kinetic and catalytic properties of the DNA (cytosine-5)-methyltransferase HhaI are described. With poly(dG-dC) as substrate, the reaction proceeds by an equilibrium (or processive) ordered Bi-Bi mechanism in which DNA binds to the enzyme first, followed by S-adenosylmethionine (AdoMet). After methyl transfer, S-adenosylhomocysteine (AdoHcy) dissociates followed by methylated DNA. AdoHcy is a potent competitive inhibitor with respect to AdoMet (Ki = 2.0 microM) and its generation during reactions results in non-linear kinetics. AdoMet and AdoHcy significantly interact with only the substrate enzyme-DNA complex; they do not bind to free enzyme and bind poorly to the methylated enzyme-DNA complex. In the absence of AdoMet, HhaI methylase catalyzes exchange of the 5-H of substrate cytosines for protons of water at about 7-fold the rate of methylation. The 5-H exchange reaction is inhibited by AdoMet or AdoHcy. In the enzyme-DNA-AdoHcy complex, AdoHcy also suppresses dissociation of DNA and reassociation of the enzyme with other substrate sequences. Our studies reveal that the catalytic mechanism of DNA (cytosine-5)-methyltransferases involves attack of the C6 of substrate cytosines by an enzyme nucleophile and formation of a transient covalent adduct. Based on precedents of other enzymes which catalyze similar reactions and the susceptibility of HhaI to inactivation by N-ethylmaleimide, we propose that the sulfhydryl group of a cysteine residue is the nucleophilic catalyst. Furthermore, we propose that Cys-81 is the active-site catalyst in HhaI. This residue is found in a Pro-Cys doublet which is conserved in all DNA (cytosine-5)-methyltransferases whose sequences have been determined to date and is found in related enzymes. Finally, we discuss the possibility that covalent adducts between C6 of pyrimidines and nucleophiles of proteins may be important general components of protein-nucleic acid interactions.     

Deleterious effects of beta-branched residues in the S1 specificity pocket of Streptomyces griseus proteinase B (SGPB): crystal structures of the turkey ovomucoid third domain variants Ile18I, Val18I, Thr18I, and Ser18I in complex with SGPB

Turkey ovomucoid third domain (OMTKY3) is a canonical inhibitor of serine proteinases. Upon complex formation, the inhibitors fully exposed P1 residue becomes fully buried in the preformed cavity of the enzyme. All 20 P1 variants of OMTKY3 have been obtained by recombinant DNA technology and their equilibrium association constants have been measured with six serine proteinases. To rationalize the trends observed in this data set, high resolution crystal structures have been determined for OMTKY3 P1 variants in complex with the bacterial serine proteinase, Streptomyces griseus proteinase B (SGPB). Four high resolution complex structures are being reported in this paper; the three beta-branched variants, Ile18I, Val18I, and Thr18I, determined to 2.1, 1.6, and 1.7 A resolution, respectively, and the structure of the Ser18I variant complex, determined to 1.9 A resolution. Models of the Cys18I, Hse18I, and Ape18I variant complexes are also discussed. The beta-branched side chains are not complementary to the shape of the S1 binding pocket in SGPB, in contrast to that of the wild-type gamma-branched P1 residue for OMTKY3, Leu18I. Chi1 angles of approximately 40 degrees are imposed on the side chains of Ile18I, Val18I, and Thr18I within the S1 pocket. Dihedral angles of +60 degrees, -60 degrees, or 180 degrees are more commonly observed but 40 degrees is not unfavorable for the beta-branched side chains. Thr18I Ogamma1 also forms a hydrogen bond with Ser195 Ogamma in this orientation. The Ser18I side chain adopts two alternate conformations within the S1 pocket of SGPB, suggesting that the side chain is not stable in either conformation.     

The methyl donor S-Adenosylmethionine inhibits active demethylation of DNA: a candidate novel mechanism for the pharmacological effects of S-Adenosylmethionine

S-Adenosylmethionine (AdoMet) is the methyl donor of numerous methylation reactions. The current model is that an increased concentration of AdoMet stimulates DNA methyltransferase reactions, triggering hypermethylation and protecting the genome against global hypomethylation, a hallmark of cancer. Using an assay of active demethylation in HEK 293 cells, we show that AdoMet inhibits active demethylation and expression of an ectopically methylated CMV-GFP (green fluorescent protein) plasmid in a dose-dependent manner. The inhibition of GFP expression is specific to methylated GFP; AdoMet does not inhibit an identical but unmethylated CMV-GFP plasmid. S-Adenosylhomocysteine (AdoHcy), the product of methyltransferase reactions utilizing AdoMet does not inhibit demethylation or expression of CMV-GFP. In vitro, AdoMet but not AdoHcy inhibits methylated DNA-binding protein 2/DNA demethylase as well as endogenous demethylase activity extracted from HEK 293, suggesting that AdoMet directly inhibits demethylase activity, and that the methyl residue on AdoMet is required for its interaction with demethylase. Taken together, our data support an alternative mechanism of action for AdoMet as an inhibitor of intracellular demethylase activity, which results in hypermethylation of DNA.     

Kinetic analysis of the histamine N-methyltransferase reaction as used in the histamine radioenzymatic assay: Optimization of assay specificity

The specificity of the histamine N-methyltransferase (HNMT) based radioenzymatic assay for histamine has been questioned since N-α-methylhistamine is also a substrate for this enzyme. Purification of HNMT for use in the radioenzymatic assay improves sensitivity and specificity of this procedure. In this investigation, further improvements in specificity, with respect to other HNMT substrates, were attained by optimization of reaction conditions based on the evaluation of HNMT kinetic parameters. These studies demonstrate that appropriate control of reaction temperature and concentration of both the enzyme and the radiolabeled methyl donor improve the specificity of this assay for histamine.     

The Ile128Thr polymorphism influences stability and ligand binding properties of the microsomal triglyceride transfer protein

The microsomal triglyceride transfer protein (MTTP) is essential for the assembly of VLDLs. We recently observed that a polymorphism in the MTTP promoter (-493G>T), which is in allelic association with an isoleucine-to-theronine substitution at position 128 (Ile128Thr) in the expressed protein, confers an increased risk of coronary heart disease. Two variant proteins comprising amino acids 16-297 of intact MTTP, MTTP(N)-Ile128 and MTTP(N)-Thr128, had similar native secondary structure content, as judged by circular dichroism. However, the thermal stability of MTTP(N)-Thr128 was greatly reduced, and this protein was also more extensively cleaved in limited proteolysis experiments compared with MTTP(N)-Ile128; both of these findings support a less compact fold. On adding LDL, which includes natively folded apolipoprotein B (apoB), decreased stability of the MTTP(N)-Thr128-LDL complex was observed compared with that of the MTTP(N)-Ile128-LDL complex. In a refined model of the N-terminal domain of MTTP, residue 128 is located in a surface-exposed position, in the same region as an identified MTTP binding site in the homologous apoB protein. Thus, the Ile128Thr polymorphism confers reduced structural stability, leading to decreased binding of MTTP to LDL particles. Because the major MTTP binding target on LDL is apoB, the Ile128Thr polymorphism could target the MTTP-apoB interaction.     

Bacteriophage T4 Dam DNA-[N6-adenine]methyltransferase. Kinetic evidence for a catalytically essential conformational change in the ternary complex.

We carried out a steady state kinetic analysis of the bacteriophage T4 DNA-[N6-adenine]methyltransferase (T4 Dam) mediated methyl group transfer reaction from S-adenosyl-l-methionine (AdoMet) to Ade in the palindromic recognition sequence, GATC, of a 20-mer oligonucleotide duplex. Product inhibition patterns were consistent with a steady state-ordered bi-bi mechanism in which the order of substrate binding and product (methylated DNA, DNA(Me) and S-adenosyl-l-homocysteine, AdoHcy) release was AdoMet downward arrow DNA downward arrow DNA(Me) upward arrow AdoHcy upward arrow. A strong reduction in the rate of methylation was observed at high concentrations of the substrate 20-mer DNA duplex. In contrast, increasing substrate AdoMet concentration led to stimulation in the reaction rate with no evidence of saturation. We propose the following model. Free T4 Dam (initially in conformational form E) randomly interacts with substrates AdoMet and DNA to form a ternary T4 Dam-AdoMet-DNA complex in which T4 Dam has isomerized to conformational state F, which is specifically adapted for catalysis. After the chemical step of methyl group transfer from AdoMet to DNA, product DNA(Me) dissociates relatively rapidly (k(off) = 1.7 x s(-1)) from the complex. In contrast, dissociation of product AdoHcy proceeds relatively slowly (k(off) = 0.018 x s(-1)), indicating that its release is the rate-limiting step, consistent with kcat = 0.015 x s(-1). After AdoHcy release, the enzyme remains in the F conformational form and is able to preferentially bind AdoMet (unlike form E, which randomly binds AdoMet and DNA), and the AdoMet-F binary complex then binds DNA to start another methylation cycle. We also propose an alternative pathway in which the release of AdoHcy is coordinated with the binding of AdoMet in a single concerted event, while T4 Dam remains in the isomerized form F. The resulting AdoMet-F binary complex then binds DNA, and another methylation reaction ensues. This route is preferred at high AdoMet concentrations.     

S-Adenosyl-l-homocysteine in brain

Administration of methionine sulfoximine (MSO) to rats and mice significantly decreased cerebral levels ofS-adenosyl-l-homocysteine (AdoHcy). Concurrent administration of methionine prevented this decrease and, when methionine was given alone, significantly elevated AdoHcy levels resulted in both species. Regionally, AdoHcy levels varied from 20 nmol/g in rat cerebellum and spinal cord to about 60 nmol/g in hypothalamus and midbrain. MSO decreased AdoHcy in all regions tested except striatum, midbrain, and spinal cord. AdoMet/AdoHcy ratios (methylation index) varied from 0.48 in hypothalamus to 2.4 in cerebellum, and MSO administration decreased these ratios in all regions except hypothalamus. AdoHcy hydrolase activity was lowest in hypothalamus, highest in brainstem and, generally, varied inversely with regional AdoHcy levels. MSO decreased AdoHcy hydrolase activity in all regions except hypothalamus and spinal cord. Cycloleucine administration resulted in significantly decreased levels of mouse brain AdoHcy, whereas the administration of dihydroxyphenylalanine (DOPA) failed to affect AdoHcy levels. It is concluded that (a) cerebral AdoHcy levels are more tightly regulated than are those of AdoMet after MSO administration, (b) slight fluctuations of AdoHcy levels may be important in regulating AdoHcy hydrolase activity and hence AdoHcy catabolism in vivo, (c) the AdoMet/AdoHcy ratio reflects the absolute AdoMet concentration rather than the transmethylation flux, (d) the decreased AdoMet levels in midbrain, cortex, and striatum after MSO with no corresponding decrease in AdoHcy suggest an enhanced AdoMet utilization, hence an increased transmethylation in the MSO preconvulsant state.Supported by USPHS, NINCDS grant NS-06294.     

S-adenosylhomocysteine metabolism in different cell lines: effect of hypoxia and cell density.

BACKGROUND/AIMS: The methylation potential (MP) is defined as the ratio of S-adenosylmethionine (AdoMet) to S-adenosylhomocysteine (AdoHcy). It was shown recently that hypoxia increases AdoMet/AdoHcy ratio in HepG2 cells (Hermes et al., Exp Cell Res 294: 325-334, 2004). In the present study, we compared AdoMet/AdoHcy ratio and energy metabolism in HepG2, HEK-293, HeLa, MCF-7 and SK-HEP-1 cell lines under normoxia and hypoxia. METHODS: Metabolite concentrations were measured by HPLC. In addition, AdoHcy hydrolase (AdoHcyase) activity was determined photometrically. RESULTS: Under normoxia HepG2 cells show the highest AdoMet/AdoHcy ratio of 53.4 +/- 3.3 followed by MCF-7 and SK-HEP-1 cells with a AdoMet/AdoHcy ratio of 14.4 +/- 1.1 and 21.1 +/- 1.3, respectively. The lowest AdoMet/AdoHcy ratios are exhibited by HeLa and HEK-293 cells (6.6 +/- 0.7 and 7.1 +/- 0.3). Hypoxia does not significantly change the MP in MCF-7 and HeLa cells, but alters the MP in HepG2, HEK-293 and SK-HEP-1 cells. These alterations are dependent on the cell density. Under normoxia HepG2 cells exhibit AdoHcyase activity of 2.5 +/- 0.2 nmol min(-1) mg(-1) protein. All other cell lines show 3-5 times lower enzyme activity. Interestingly, hypoxia affects AdoHcyase activity only in HepG2 cells. CONCLUSIONS: Our data clearly show that the cell lines are characterized by different MP and different behavior under hypoxia. That implies that a lower MP is not necessarily associated with impaired transmethylation activity and cellular function.     

Biosynthesis of phytosterols. Kinetic mechanism for the enzymatic C-methylation of sterols

Cloned soybean sterol methyltransferase was purified from Escherichia coli to gel electrophoretic homogeneity. From initial velocity experiments, catalytic constants for substrates best suited for the first and second C1 transfer activities, cycloartenol and 24(28)-methylenelophenol, were 0.01 and 0.001 s-1, respectively. Two-substrate kinetic analysis using cycloartenol and S-adenosyl-l-methionine (AdoMet) generated an intersecting line pattern characteristic of a ternary complex kinetic mechanism. The high energy intermediate analog 25-azacycloartanol was a noncompetitive inhibitor versus cycloartenol and an uncompetitive inhibitor versus AdoMet. The dead end inhibitor analog cyclolaudenol was competitive versus cycloartenol and uncompetitive versus AdoMet. 24(28)-Methylenecycloartanol and AdoHcy generated competitive and noncompetitive kinetic patterns, respectively, with respect to AdoMet. Therefore, 24(28)-methylenecycloartanol combines with the same enzyme form as does cycloartenol and must be released from the enzyme before AdoHcy. 25-Azacycloartanol inhibited the first and second C1 transfer activities with about equal efficacy (Ki = 45 nm), suggesting that the successive C-methylation of the Delta 24 bond occurs at the same active center. Comparison of the initial velocity data using AdoMet versus [2H3-methyl]AdoMet as substrates tested against saturating amounts of cycloartenol indicated an isotope effect on VCH3/VCD3 close to unity. [25-2H]24(28)-Methylenecycloartanol, [28E-2H]24 (28)-methylenelanosterol, and [28Z-2H]24(28)-methylene lanosterol were prepared and paired with AdoMet or [methyl-3H3]AdoMet to examine the kinetic isotope effects attending the C-28 deprotonation in the enzymatic synthesis of 24-ethyl(idene) sterols. The stereochemical features as well as the observation of isotopically sensitive branching during the second C-methylation suggests that the two methylation steps can proceed by a change in chemical mechanism resulting from differences in sterol structure, concerted versus carbocation; the kinetic mechanism remains the same during the consecutive methylation of the Delta 24 bond.     

Sources of S-adenosyl-l-homocysteine background in measuring protein arginine N-methyltransferase activity using tandem mass spectrometry

S-Adenosyl-l-homocysteine (AdoHcy) background signal in reactions with protein arginine N-methyltransferase 1 is investigated using an ultrahigh-performance liquid chromatography tandem mass spectrometry assay that measures AdoHcy. We identify three sources of AdoHcy background: enzymatic automethylation, AdoHcy contamination in commercial S-adenosyl-l-methionine (AdoMet), and nonenzymatic pseudo-first-order formation of AdoHcy from AdoMet. We propose a potential mechanism for the nonenzymatic production of AdoHcy and illustrate strategies for mitigating background AdoHcy that can be applied to any assay.