ABSTRACT
Betaine aldehyde levels were determined in rat livers following 4 weeks of ethanol feeding, employing the Lieber-De Carli liquid diet. The results showed that the levels of betaine aldehyde are unaffected by alcohol feeding to rats. These levels in both experimental and control animals were found to be quite low, 5.5 nmol/g liver. Betaine aldehyde levels have not been determined previously in mammalian liver because of methodological difficulties. This investigation employed fast atom bombardment-mass spectroscopy to determine the levels of betaine aldehyde, betaine, and choline. The decrease in betaine levels following ethanol administration confirmed the results of other investigators. Choline levels determined during this investigation were lower than previously reported. The reason for starting this investigation was the fact that the enzyme that catalyzes betaine aldehyde dehydrogenation to betaine, which is distributed in both mitochondria and the cytoplasm, was found to also metabolize acetaldehyde with K(m) and V(max) values lower than those for betaine aldehyde. Thus, it appeared likely that the metabolism of acetaldehyde during ethanol metabolism might inhibit betaine aldehyde conversion to betaine and thereby result in decreased betaine levels (Barak et al., Alcohol 13: 395-398, 1996). The fact that betaine aldehyde levels in alcohol-fed animals were similar to those in controls demonstrates that competition between acetaldehyde and betaine aldehyde for the same enzyme does not occur. This complete lack of competition suggests that betaine aldehyde dehydrogenase in the mitochondrial matrix may totally metabolize betaine aldehyde to betaine without any involvement of cytoplasmic betaine aldehyde dehydrogenase.
Subject(s)
Betaine/analogs & derivatives , Betaine/metabolism , Choline/metabolism , Ethanol/metabolism , Liver/metabolism , Acetaldehyde/chemistry , Animals , Betaine/chemistry , Central Nervous System Depressants/pharmacology , Cytoplasm/drug effects , Cytoplasm/metabolism , Ethanol/pharmacology , Isoelectric Focusing , Kinetics , Liver/drug effects , Male , Mitochondria, Liver/drug effects , Mitochondria, Liver/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
We investigated the epoxidase activity of a class mu glutathione S-transferase (cGSTM1-1), using 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) as substrate. Trp209 on the C-terminal tail, Arg107 on the alpha4 helix, Asp161 and Gln165 on the alpha6 helix of cGSTM1-1 were selected for mutagenesis and kinetic studies. A hydrophobic side-chain at residue 209 is needed for the epoxidase activity of cGSTM1-1. Replacing Trp209 with histidine, isoleucine or proline resulted in a fivefold to 28-fold decrease in the k(cat)(app) of the enzyme, while a modest 25 % decrease in the k(cat)(app) was observed for the W209F mutant. The rGSTM1-1 enzyme has serine at the correponding position. The k(cat)(app) of the S209W mutant is 2. 5-fold higher than that of the wild-type rGSTM1-1. A charged residue is needed at position 107 of cGSTM1-1. The K(m)(app)(GSH) of the R107L mutant is 38-fold lower than that of the wild-type enzyme. On the contrary, the R107E mutant has a K(m)(app)(GSH) and a k(cat)(app) that are 11-fold and 35 % lower than those of the wild-type cGSTM1-1. The substitutions of Gln165 with Glu or Leu have minimal effect on the affinity of the mutants towards GSH or EPNP. However, a discernible reduction in k(cat)(app) was observed. Asp161 is involved in maintaining the structural integrity of the enzyme. The K(m)(app)(GSH) of the D161L mutant is 616-fold higher than that of the wild-type enzyme. In the hydrogen/deuterium exchange experiments, this mutant has the highest level of deuteration among all the proteins tested. We also elucidated the structure of cGSTM1-1 co-crystallized with the glutathionyl-conjugated 1, 2-epoxy-3-(p-nitrophenoxy)propane (EPNP) at 2.8 A resolution. The product found in the active site was 1-hydroxy-2-(S-glutathionyl)-3-(p-nitrophenoxy)propane, instead of the conventional 2-hydroxy isomer. The EPNP moiety orients towards Arg107 and Gln165 in dimer AB, and protrudes into a hydrophobic region formed by the loop connecting beta1 and alpha1 and part of the C-terminal tail in dimer CD. The phenoxyl ring forms strong ring stacking with the Trp209 side-chain in dimer CD. We hypothesize that these two conformations represent the EPNP moiety close to the initial and final stages of the reaction mechanism, respectively.
Subject(s)
Amino Acid Substitution/genetics , Glutathione Transferase/chemistry , Glutathione Transferase/metabolism , Nitrophenols/metabolism , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Animals , Binding Sites , Chickens , Crystallography, X-Ray , Deuterium/metabolism , Epoxy Compounds/chemistry , Epoxy Compounds/metabolism , Glutamine/genetics , Glutamine/metabolism , Glutathione/metabolism , Glutathione Transferase/classification , Glutathione Transferase/genetics , Hydrogen Bonding , Kinetics , Models, Molecular , Mutation/genetics , Nitrophenols/chemistry , Oxidoreductases/classification , Oxidoreductases/genetics , Protein Conformation , Protons , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Structure-Activity Relationship , Temperature , Tryptophan/genetics , Tryptophan/metabolism , Tyrosine/genetics , Tyrosine/metabolismABSTRACT
Betaine aldehyde dehydrogenase has been purified to homogeneity from rat liver mitochondria. The properties of betaine aldehyde dehydrogenase were similar to those of human cytoplasmic E3 isozyme in substrate specificity and kinetic constants for substrates. The primary structure of four tryptic peptides was also similar; only two substitutions, at most, per peptide were observed. Thus, betaine aldehyde dehydrogenase is not a specific enzyme, as formerly believed; activity with betaine aldehyde is a property of aldehyde dehydrogenase (EC 1.2.1.3), which has broad substrate specificity. Up to the present time the enzyme was thought to be cytoplasmic in mammals. This report establishes, for the first time, mitochondrial subcellular localization for aldehyde dehydrogenase, which dehydrogenates betaine aldehyde, and its colocalization with choline dehydrogenase. Betaine aldehyde dehydrogenation is an important function in the metabolism of choline to betaine, a major osmolyte. Betaine is also important in mammalian organisms as a major methyl group donor and nitrogen source. This is the first purification and characterization of mitochondrial betaine aldehyde dehydrogenase from any mammalian species.
Subject(s)
Aldehyde Oxidoreductases/metabolism , Cytoplasm/enzymology , Isoenzymes/metabolism , Mitochondria, Liver/enzymology , Aldehyde Oxidoreductases/chemistry , Aldehyde Oxidoreductases/isolation & purification , Amino Acid Sequence , Animals , Betaine-Aldehyde Dehydrogenase , Chromatography, Liquid , Electrophoresis, Polyacrylamide Gel , Humans , Isoelectric Focusing , Isoenzymes/chemistry , Kinetics , Male , Molecular Sequence Data , Molecular Weight , Rats , Rats, Sprague-Dawley , Sequence Homology, Amino AcidSubject(s)
Aldehyde Dehydrogenase/chemistry , Isoenzymes/chemistry , Aldehyde Dehydrogenase/genetics , Amino Acid Sequence , Animals , Humans , Isoenzymes/genetics , Molecular Sequence Data , Open Reading Frames , Peptide Fragments/chemistry , Rats , Reading Frames , Sequence Alignment , Sequence Homology, Amino AcidSubject(s)
Aldehyde Dehydrogenase/metabolism , Isoenzymes/metabolism , Aldehyde Dehydrogenase/chemistry , Aldehyde Dehydrogenase/isolation & purification , Amino Acid Sequence , Betaine/analogs & derivatives , Betaine/metabolism , Brain/enzymology , Chromatography, Affinity , Chromatography, Ion Exchange , Cloning, Molecular , Enzyme Stability , Humans , Isoenzymes/chemistry , Isoenzymes/isolation & purification , Liver/enzymology , Molecular Sequence Data , Organ Specificity , Peptide Fragments/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Substrate SpecificityABSTRACT
The E3 isozyme of human aldehyde dehydrogenase (EC 1.2.1.3), with broad substrate specificity, which also catalyzes dehydrogenation of 4-aminobutyraldehyde, was purified and sequenced recently (1,3). It has been shown during this investigation to have betaine aldehyde dehydrogenase activity. Betaine aldehyde and 4-aminobutyraldehyde activities copurified on six chromatographic columns. Molecular properties of the homogeneous product were identical with those of E3 isozyme. Activity with betaine aldehyde was considerably higher than that with 4-aminobutyraldehyde, the best known substrate. Thus, human E3 isozyme and betaine aldehyde dehydrogenase (EC 1.2.1.8) are the same enzyme.
