Properties of α-Amino-ε-caprolactam Racemase from Achromobacter obae

α-Amino-ε-caprolactam racemase, which occurs in the cytoplasmic fraction of Achromobacter obae, has been purified to homogeneity. It has a monomeric structure with a molecular weight of approximately 50,000. The absorption spectrum of the enzyme exhibits maxima at 280 and 412 nm at pH 7.3, and is independent of pH from 6.0 to 8.0. One mole of pyridoxal 5′-phosphate is bound per mol of the enzyme. Incubation of the enzyme with hydroxylamine resulted in the formation of the apoenzyme. d- and l-α-Amino-ε-caprolactams are the only substrates. The maximum activity is found at pH 8.8 for both the isomers. Michaelis constants are as follows: 8 mm for d-α-amino-ε-caprolactam, 6mm for l-α-amino-ε-caprolactam and 2.1 × 10^?7 m for pyridoxal 5′-phosphate. The enzyme is inhibited significantly by CuSO₄, HgCl₂, thiol reagents such as N-ethylmaleimide and p-chloromercuribenzoate, and carbonyl reagents (e.g., phenylhydrazine and hydroxylamine). α-Amino-ε-caprolactam racemase catalyzes the α-proton exchange of the substrate with deuteron during racemization in deuterium oxide.     

Bacterial Racemization of α-Amino-ε-caprolactam

1. Several bacteria were isolated from soil which grew on both d- and l-aminolactam and whose cells had an activity to racemize them. They were identified as Achromobacter obae nov. sp., Achr. cycloclastes, Alcaligenes faecalis and Flavobacterium arborescens.

2. Racemization of d- and l-aminolactam was investigated using the lyophilized cells of Achr. obae nov. sp. The optimum pH value of the reaction was about 8.0. The racemizing activity was completely inhibited by 10^?4 m hydroxylamine, and the inhibition was removed by 10^?4 m pyridoxal phosphate. Five percent d- and l-aminolactam solutions were completely racemized with a concomitant slight formation of l-lysine.     

The Mechanism of Inactivation of Bacteriophage ϕX174 by Autoxidizable Synthetic Polysaccharides

Autoxidizable synthetic polysaccharides prepared by polycondensation of reducing aldose or ketose in dimethyl sulfoxide containing pohsphorus pentaoxide [Polymer, 13, 190 (1972)] inactivated phage ?X174. Another autoxidizable polysaccharides obtained by oxidation of natural glucans with the same oxidant also inactivated ?X174. The ?X174 inactivation was due to strand scission of viral DNA in the virion. The inactivation reaction was stimulated by Cu²⁺ and inhibited by EDTA, Superoxide dismutase, catalase and several radical scavengers. These results suggest that oxygen radicals produced during autoxidation of polysaccharides are responsible for ?X174 inactivation.     

Inactivation of peptidylglycine α-hydroxylating monooxygenase by cinnamic acid analogs

Peptidylglycine α-amidating monooxygenase (PAM) is a bifunctional enzyme that catalyzes the final reaction in the maturation of α-amidated peptide hormones. Peptidylglycine α-hydroxylating monooxygenase (PHM) is the PAM domain responsible for the copper-, ascorbate- and O₂-dependent hydroxylation of a glycine-extended peptide. Peptidylamidoglycolate lyase is the PAM domain responsible for the Zn(II)-dependent dealkylation of the α-hydroxyglycine-containing precursor to the final α-amidated peptide. We report herein that cinnamic acid and cinnamic acid analogs are inhibitors or inactivators of PHM. The inactivation chemistry exhibited by the cinnamates exhibits all the attributes of a suicide-substrate. However, we find no evidence for the formation of an irreversible linkage between cinnamate and PHM in the inactivated enzyme. Our data support the reversible formation of a Michael adduct between an active site nucleophile and cinnamate that leads to inactive enzyme. Our data are of significance given that cinnamates are found in foods, perfumes, cosmetics and pharmaceuticals.     

Irreversible Inactivation of l-Ornithine : α-Ketoglutarate δ-Aminotransferase by Gabaculine

The action of endo-1,4-β-d-galactanase from Penicillium citrinum on o-nitrophenyl-β-d-galactopyranoside (ONPG) was investigated.

In reaction mixtures with various concentrations of ONPG, liberation of o-nitrophenol (ONP) was observed after a lag phase and then oligosaccharides with and without ONP-group were found to accumulate. The products were separated by activated carbon column chromatography and paper chromatography, and found to be a series of β-1,4-linked galactooligosaccharides and their ONP-substituted derivatives.

Liberation of ONP from the ONP-substituted oligosaccharides by the enzyme occured without a lag phase. Furthermore, the lag phase of ONP liberation from ONPG was eliminated by the addition of β-1,4-galactotriose and -tetraose to the reaction mixture.

The formation of ONP-substituted oligosaccharides before the liberation of ONP is assumed to be the cause of the observed lag.     

Inactivation and structural alteration of α-amylase by low-pressure carbon dioxide microbubbles

The efficiency of low-pressure carbon dioxide microbubbles (CO₂MB) to inactivate α-amylase was analysed kinetically, and structural alteration of α-amylase by CO₂MB was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorescence analysis of tryptophan (Trp) residues. Activity and Trp fluorescence intensity of α-amylase treated by CO₂MB decreased with increasing temperature, pressure and exposure time, and lowering the initial buffer pH, respectively. In the kinetic analysis, it was confirmed that the decreased temperature-dependency and increased activation energy associated with the inactivation of α-amylase by CO₂MB were induced by pressurizing the mixing vessel and that the decreased pressure-dependency and increased activation volume concomitant to the inactivation of α-amylase by CO₂MB was induced by increasing the temperature in the heating coil. In SDS-PAGE, CO₂MB was suggested to induce the structural alteration of α-amylase because the band density decreased after CO₂MB treatment, although this phenomenon was not related to the inactivation efficiency. However, Trp fluorescence analysis showed that the alteration of the tertiary structure of α-amylase by CO₂MB was related to the inactivation efficiency. Therefore, CO₂MB was more effective than thermal treatment in inactivating α-amylase, and the inactivation efficiency was suggested to be related to the alteration of the enzyme’s tertiary structure.     

Mechanism of action of α-galactosidase

The effect ofpH on K_m and V_max values of coconut α-galactosidase indicates the involvement of two ionizing groups with pK_a values of 3.5 and 6.5 in catalysis. Chemical modification has indicated the presence of two carboxyl groups, a tryptophan and a tyrosine, at or near the active site of α-galactosidase. Based on these facts a new mechanism of action for α-galactosidase is proposed in which the ionizing group with a pK_a of 3.5 is a carboxyl group involved in stabilizing a carbonium ion intermediate and the ionizing group with a pK_a of 6.5 is a carboxyl group perturbed due to the presence of a hydrophobic residues in its vicinity which donates a H⁺ ion in catalysis.     

Inactivation of Bacteriophages by ε-Poly-l-lysine Produced by Streptomyces

Degradation of a β-O-4lignin substructure model dimer by a white rot fungus, Phanerochaete chrysosporium, was investigated using a culture containing H₂¹⁸O, and the following conclusions were made. a) The direct hydrolysis at C_β of the β-O-4 dimer was not involved in formation of arylglycerol. b) About half of the oxygen at the benzyl (C_α) position of the glycerol was derived from H₂O (H₂¹⁸O) and the other half was from the oxygen at the benzyl (C_α) position of the substrate β-O-4 dimer. c) But, the oxygen at the C_α position of the substrate β-O-4 dimer did not migrate to the C_α position of the aryglycerol.     

Membrane Permeabilization by Oligomeric α-Synuclein: In Search of the Mechanism

Background

The question of how the aggregation of the neuronal protein α-synuclein contributes to neuronal toxicity in Parkinson''s disease has been the subject of intensive research over the past decade. Recently, attention has shifted from the amyloid fibrils to soluble oligomeric intermediates in the α-synuclein aggregation process. These oligomers are hypothesized to be cytotoxic and to permeabilize cellular membranes, possibly by forming pore-like complexes in the bilayer. Although the subject of α-synuclein oligomer-membrane interactions has attracted much attention, there is only limited evidence that supports the pore formation by α-synuclein oligomers. In addition the existing data are contradictory.

Methodology/Principal Findings

Here we have studied the mechanism of lipid bilayer disruption by a well-characterized α-synuclein oligomer species in detail using a number of in vitro bilayer systems and assays. Dye efflux from vesicles induced by oligomeric α-synuclein was found to be a fast all-or-none process. Individual vesicles swiftly lose their contents but overall vesicle morphology remains unaltered. A newly developed assay based on a dextran-coupled dye showed that non-equilibrium processes dominate the disruption of the vesicles. The membrane is highly permeable to solute influx directly after oligomer addition, after which membrane integrity is partly restored. The permeabilization of the membrane is possibly related to the intrinsic instability of the bilayer. Vesicles composed of negatively charged lipids, which are generally used for measuring α-synuclein-lipid interactions, were unstable to protein adsorption in general.

Conclusions/Significance

The dye efflux from negatively charged vesicles upon addition of α-synuclein has been hypothesized to occur through the formation of oligomeric membrane pores. However, our results show that the dye efflux characteristics are consistent with bilayer defects caused by membrane instability. These data shed new insights into potential mechanisms of toxicity of oligomeric α-synuclein species.     

Mechanism and stereochemistry of α,β-elimination of l-tyrosine catalysed by tyrosine phenol-lyase

The decomposition of l-tyrosine and its α-deuterated analogue under the action of extracts from Escherichia intermedia A-21 with high tyrosine phenol-lyase [l-tyrosine phenol-lyase (deaminating), EC 4.1.99.2] activity has been studied. The mass spectrometric data for samples of phenol produced by decomposition of deutero-l-tyrosine in water and D₂O-water (10:1) mixture, and by decomposition of normal l-tyrosine in D₂O-water (10:1), show that the process is accompanied by the intramolecular transfer of D or H to the leaving phenol group. The degree of transfer is 7–10%. Thus, the abstraction of α-proton and the subsequent protonation of the aromatic ring are accomplished by the same functional group of the enzyme. This is indicative of the cis-orientation of α-proton and the phenol fragment in relation to the plane of Schiff's base of α-aminoacrylate with pyridoxal phosphate during α,β-elimination. The isotope effect of the studied enzymic reaction is ≈3, which allows us to consider the α-proton abstraction as the limiting stage of the process.     

Mechanism of the cyanide-catalyzed oxidation of α-ketoaldehydes and α-ketoalcohols

Cyanide catalyzes the reduction of dioxygen or of ferricytochrome c by dihydroxyacetone phosphate. The rapid initial phase of these reactions, but not the subsequent slow phase, was augmented by incubating the triose phosphate aerobically or anaerobically at pH 9.0 prior to adding the cyanide. The aerobic incubation, which was most effective, was associated with a decline in enediol, whereas the less effective anaerobic incubation was accompanied by an increase in enediol content. This suggested that the α-ketoaldehyde product of autoxidation of the enediol, rather than the enediol itself, was responsible for the rapid phase reaction which followed addition of cyanide. This was confirmed by exploring the cyanide-catalyzed oxidation of the α-ketoaldehyde, phenylglyoxal. The inhibitory effect of the manganese-containing superoxide dismutase indicated that O₂⁻ was a kinetically important intermediate of the rapid phase reaction. A reaction mechanism is proposed which is consistent with the results presented.     

Effects of Various Dietary Fatty Acids on α-Amino-β-carboxymuconate-ε-semialdehyde Decarboxylase Activity in Rat Liver

In this study, we investigated the effects of short-chain, middle-chain, and long-chain fatty acids on the activity of rat liver α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase [EC 4.1.1.45] (ACMSD), a key enzyme of tryptophan-niacin metabolism. Moreover, we examined the cholesterol metabolism and lipid peroxidation in relation to ACMSD activity in rats. When diets containing 2%, 5%, and 10% levels of fatty acids were given to rats for a week, saturated fatty acids and elaidic acid (trans form) did not suppress the ACMSD activity in liver. But polyunsaturated fatty acids such as linoleic acid, linolenic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) strongly suppressed the liver ACMSD activity. Five % sorbic acid and oleic acid tended to suppress the liver ACMSD activity weakly. On the other hand, this report indicated that there is no correlation between liver ACMSD activity and cholesterol levels of serum or liver, but there is a weak negative correlation between liver malondialdehyde concentration and liver ACMSD specific activity.     

Mechanism of Deep-Sea Fish α-Actin Pressure Tolerance Investigated by Molecular Dynamics Simulations

The pressure tolerance of monomeric α-actin proteins from the deep-sea fish Coryphaenoides armatus and C. yaquinae was compared to that of non-deep-sea fish C. acrolepis, carp, and rabbit/human/chicken actins using molecular dynamics simulations at 0.1 and 60 MPa. The amino acid sequences of actins are highly conserved across a variety of species. The actins from C. armatus and C. yaquinae have the specific substitutions Q137K/V54A and Q137K/L67P, respectively, relative to C. acrolepis, and are pressure tolerant to depths of at least 6000 m. At high pressure, we observed significant changes in the salt bridge patterns in deep-sea fish actins, and these changes are expected to stabilize ATP binding and subdomain arrangement. Salt bridges between ATP and K137, formed in deep-sea fish actins, are expected to stabilize ATP binding even at high pressure. At high pressure, deep-sea fish actins also formed a greater total number of salt bridges than non-deep-sea fish actins owing to the formation of inter-helix/strand and inter-subdomain salt bridges. Free energy analysis suggests that deep-sea fish actins are stabilized to a greater degree by the conformational energy decrease associated with pressure effect.     

Inactivation of dopamine β-monooxygenase by hydrogen peroxide and by ascorbate

The inactivation of the water-soluble form of bovine adrenal dopamine β-monooxygenase by H₂O₂ and by ascorbate was studied. Inactivation by H₂O₂ was slow for the copper-free apoenzyme, but addition of copper gave a rapid inactivation. The results presented indicate that the enzyme-bound copper during this inactivation catalyzes partial destruction of its own binding site. The reaction orders for the inactivation by H₂O₂ seem to be 1.0 with respect to the enzyme and in the range 0.6 to 0.8 with respect to H₂O₂. The rate of inactivation obtained in the presence of ascorbate increases with addition of copper and is faster than that obtained by similar concentrations of H₂O₂. The data could not, however, be used to decide whether the inactivation by ascorbate was catalyzed by the enzymebound copper. The inactivation reaction in the presence of ascorbate seems to be of first order with respect to ascorbate at ascorbate concentrations less than 40 μm and decreases toward zero as the ascorbate concentration is increased. Experiments with the Cu(I)-chelator, bathocuproine disulfonate, revealed that inactivation led to weaker binding of copper to the protein, and this effect was more pronounced with H₂O₂ than with ascorbate.     

Mechanism of selection of side-chain rotamers in α-helices

In this study, a possible mechanism of selection of side-chain rotamers based on the rotamer distributions in known coiled-coil proteins is suggested. According to this mechanism, interhelical hydrophobic, polar, and packing interactions bring alpha-helices closer to each other and this effect squeezes side chains out of the helix-helix interface. As a result, in dimeric coiled coils and long alpha-alpha-hairpins where alpha-helices are packed in a face-to-face manner, most side chains occupying the a-positions have t-rotamers and those in the d-positions g(-)-rotamers. In tetramers, where alpha-helices are packed side-by-side, most side chains in the a-positions adopt g(-)-rotamers and those in the d-positions t-rotamers.     

Inactivation and reactivation of the mitochondrial α-ketoglutarate dehydrogenase complex

Reduced brain metabolism is an invariant feature of Alzheimer Disease (AD) that is highly correlated to the decline in brain functions. Decreased activities of key tricarboxylic acid cycle (TCA) cycle enzymes may underlie this abnormality and are highly correlated to the clinical state of the patient. The activity of the α-ketoglutarate dehydrogenase complex (KGDHC), an arguably rate-limiting enzyme of the TCA cycle, declines with AD, but the mechanism of inactivation and whether it can be reversed remains unknown. KGDHC consists of multiple copies of three subunits. KGDHC is sensitive to oxidative stress, which is pervasive in AD brain. The present studies tested the mechanism for the peroxynitrite-induced inactivation and subsequent reactivation of purified and cellular KGDHC. Peroxynitrite inhibited purified KGDHC activity in a dose-dependent manner and reduced subunit immunoreactivity and increased nitrotyrosine immunoreactivity. Nano-LC-MS/MS showed that the inactivation was related to nitration of specific tyrosine residues in the three subunits. GSH diminished the nitrotyrosine immunoreactivity of peroxynitrite-treated KGDHC, restored the activity and the immunoreactivity for KGDHC. Nano-LC-MS/MS showed this was related to de-nitration of specific tyrosine residues, suggesting KGDHC may have a denitrase activity. Treatment of N2a cells with peroxynitrite for 5 min followed by recovery of cells for 24 h reduced KGDHC activity and increased nitrotyrosine immunoreactivity. Increasing cellular GSH in peroxynitrite-treated cells rescued KGDHC activity to the control level. The results suggest that restoring KGDHC activity is possible and may be a useful therapeutic approach in neurodegenerative diseases.     

Effects of Dietary Casein and Adrenal Hormone on the Dietary Induction of α-Amino-β-carboxymuconate-ε-semialdehyde Decarboxylase Activity in Rat

The effects of dietary casein and adrenal hormone on the dietary induction of α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSDase) activity in rat liver and kidneys were examined. Of three levels of casein in the diet examined (7.5%, 15% and 30%), only the feeding of a 30% casein diet to normal rats after three days on a protein-free diet resulted in a significant increase in the activity of ACMSDase in liver and kidneys on the following morning. The degree of the increase in this activity was higher in the liver than the kidneys. Dietary induction of ACMSDase activity disappeared when rats were adrenalectomized six days prior to the start of the experiment. In sham-operated rats, dietary induction of liver ACMSDase activity was as high as that in normal rats, however, that in kidneys disappeared. Prednisolone (synthetic adrenal hormone with glucocorticoid activity) injection of adrenalectomized rats led to the recovery of the dietary induction of liver ACMSDase activity. Blood serum corticosterone levels in normal rats on the last day of the feeding period remained maximum and then decreased gradually until 04:00, and tended to be higher in rats fed a protein-free diet than in those fed a 30% casein diet. These results suggest that the dietary induction of ACMSDase activity occurs only when a sufficient amount of dietary casein is ingested in the presence of a physiologically significant concentration of blood serum glucocorticoid in rats.