Identical Subunits Of Molecular Mass Research Articles

The Iron-Sulfur Cluster-free Hydrogenase (Hmd) Is a Metalloenzyme with a Novel Iron Binding Motif

The iron-sulfur cluster-free hydrogenase (Hmd) from methanogenic archaea harbors an iron-containing cofactor of yet unknown structure. X-ray absorption spectroscopy of the active, as isolated enzyme from Methanothermobacter marburgensis (mHmd) and of the active, reconstituted enzyme from Methanocaldococcus jannaschii (jHmd) revealed the presence of mononuclear iron with two CO, one sulfur and one or two N/O in coordination distance. In jHmd, the single sulfur ligand is most probably provided by Cys176, as deduced from a comparison of the activity and of the x-ray absorption and Mössbauer spectra of the enzyme mutated in any of the three conserved cysteines. In the isolated Hmd cofactor, two CO, one sulfur, and two nitrogen/oxygen atoms coordinate the iron, the sulfur ligand being most probably provided by mercaptoethanol, which is absolutely required for the extraction of the iron-containing cofactor from the holoenzyme and for the stabilization of the extracted cofactor. In active mHmd holoenzyme, the number of iron ligands increased by one when one of the Hmd inhibitors (CO or KCN) were present, indicating that in active Hmd, the iron contains an open coordination site, which is proposed to be the site of H2 interaction.

Purification and characterisation of xylitol dehydrogenase from Neurospora crassa NCL communication No. 6347.

The purification and characterisation of one of the key enzymes in the conversion of xylose to ethanol, xylitol dehydrogenase (XD), from Neurospora crassa is reported. This organism has the unique ability to directly convert biomass to ethanol. The enzyme was purified 81-fold by fractional ammonium sulfate precipitation, column chromatography on DEAE-cellulose, Sephacryl S-200 and β-NAD agarose. The purified enzyme had a native molecular mass of 87 kDa (gel filtration) and was composed of two identical subunits of molecular mass 43.6 kDa (SDS-PAGE). The enzyme was most active at pH 8.4 and at 28°C. It was most stable at pH 7 and at 4°C. It was activated by Mg2+ and stabilised by Ca2+, Mg2+ and Mn2+. It showed activity towards xylitol and sorbitol, the respective Kms being 28.5 and 100 mM and the Km for NAD was 0.7 mM.

Hyperthermophilic and salt-dependent formyltransferase from Methanopyrus kandleri.

Methanopyrus kandleri is a hyperthermophilic methanogenic archaeon, which grows on H(2) and CO(2) as its sole energy source. Its growth temperature optimum is 98 degrees C. One of the interesting characteristics of this archaeon is its high intracellular salt content. The organism has been reported to contain the trianionic cDPG (cyclic 2,3-diphosphoglycerate) and K+ at concentrations of 1.1 and 3 M, respectively. Reflecting the high cellular salt concentration, the enzymes in this organism are adapted not only to high temperature but also to high salt concentrations. The formyltransferase from M. kandleri was characterized extensively with respect to thermo- and halophilicity. The crystal structure of the formyltransferase at 1.73 A shows the enzyme to be composed of four identical subunits of molecular mass 32 kDa. The formyltransferase is thermostable and active only at relatively high concentrations of potassium phosphate (1 M) or other salts with strongly hydrated anions (strong salting-out salts). Potassium phosphate and potassium cDPG were found to be equivalent in activating and stabilizing the enzyme. At low concentrations of these salts, the enzyme is inactive and thermolabile. It was shown by equilibrium sedimentation analysis that the enzyme is in a monomer/dimer/tetramer equilibrium, the equilibrium constant being dependent on the concentration of salts: the higher oligomeric species increase with increasing salt concentrations. Evidence was provided that the monomer is both inactive and thermolabile. Experiments using a mutation which is directed to break surface ion pairs between two dimers indicated that dimerization is required for activity and tetramerization leads to thermostability.

Isolation, purification, and physicochemical characterization of a d-galactose-binding lectin from seeds of Erythrina speciosa

A lectin was isolated from the saline extract of Erythrina speciosa seeds by affinity chromatography on lactose–Sepharose. The lectin content was about 265 mg/100 g dry flour. E. speciosa seed lectin (EspecL) agglutinated all human RBC types, showing no human blood group specificity; however a slight preference toward the O blood group was evident. The lectin also agglutinated rabbit, sheep, and mouse blood cells and showed no effect on horse erythrocytes. Lactose was the most potent inhibitor of EspecL hemagglutinating activity (minimal inhibitory concentration (MIC)=0.25 mM) followed by N-acetyllactosamine, MIC=0.5 mM, and then p-nitrophenyl α-galactopyranoside, MIC=2 mM. The lectin was a glycoprotein with a neutral carbohydrate content of 5.5% and had two p I values of 5.8 and 6.1 and E 1% 1 cm of 14.5. The native molecular mass of the lectin detected by hydrodynamic light scattering was 58 kDa and when examined by mass spectroscopy and SDS–PAGE it was found to be composed of two identical subunits of molecular mass of 27.6 kDa. The amino acid composition of the lectin revealed that it was rich in acidic and hydroxyl amino acids, contained a lesser amount of methionine, and totally lacked cysteine. The N-terminal of the lectin shared major similarities with other reported Erythrina lectins. The lectin was a metaloprotein that needed both Ca 2+ and Mn 2+ ions for its activity. Removal of these metals by EDTA rendered the lectin inactive whereas their addition restored the activity. EspecL was acidic pH sensitive and totally lost its activity when incubated with all pH values between pH 3 and pH 6. Above pH 6 and to pH 9.6 there was no effect on the lectin activity. At 65 °C for more than 90 min the lectin was fairly stable; however, when heated at 70 °C for 10 min it lost more than 80% of its original activity and was totally inactivated at 80 °C for less than 10 min. Fluorescence studies of EspecL indicated that tryptophan residues were present in a highly hydrophobic environment, and binding of lactose to EspecL neither quenched tryptophan fluorescence nor altered λ max position. Treating purified EspecL with NBS an affinity-modifying reagent specific for tryptophan totally inactivated the lectin with total modification of three tryptophan residues. Of these residues only the third modified residue seemed to play a crucial role in the lectin activity. Addition of lactose to the assay medium did not provide protection against NBS modification which indicated that tryptophan might not be directly involved in the binding of haptenic sugar d-galactose. Modification of tyrosine with N-acetylimidazole led to a 50% drop in EspecL activity with concomitant acetylation of six tyrosine residues. The secondary structure of EspecL as studied by circular dichroism was found to be a typical β-pleated-sheet structure which is comparable to the CD structure of Erythrina corallodendron lectin. Binding of lactose did not alter the EspecL secondary structure as revealed by CD examination.

Biotransformation of crotonobetaine to L(-)-carnitine in Proteus sp.

Two proteins, component I (CI) and component II (CII), catalyze the biotransformation of crotonobetaine to L(-)-carnitine in Proteus sp. CI was purified to electrophoretic homogeneity from cell-free extracts of Proteus sp. The N-terminal amino acid sequence of CI showed high similarity (80%) to the caiB gene product from Escherichia coli O44K74, which encodes the L(-)-carnitine dehydratase. CI alone was unable to convert crotonobetaine into L(-)-carnitine even in the presence of the cosubstrates crotonobetainyl-CoA or gamma-butyrobetainyl-CoA, which are essential for this biotransformation. The relative molecular mass of CI was determined to be 91.1 kDa. CI is composed of two identical subunits of molecular mass 43.6 kDa. The isoelectric point is 5.0. CII was purified to electrophoretic homogeneity from cell-free extracts of Proteus sp. and its N-terminal amino acid sequence showed high similarity (75%) to the caiD gene product of E. coli O44K74. The relative molecular mass of CII was shown to be 88.0 kDa, and CII is composed of three identical subunits of molecular mass 30.1 kDa. The isoelectric point of CII is 4.9. For the biotransformation of crotonobetaine to L(-)-carnitine, the presence of CI, CII, and a cosubstrate (crotonobetainyl-CoA or gamma-butyrobetainyl-CoA) were shown to be essential.

Purification and characterization of tyrosine decarboxylase of Lactobacillus brevis IOEB 9809 isolated from wine

Tyrosine decarboxylase (EC 4.1.1.25) (TDC) from the wine Lactobacillus brevis IOEB 9809 was purified by a rapid procedure involving anion exchange chromatography, ultrafiltration and hydrophobic interaction chromatography. The protein comprised two subunits of identical molecular mass (approximately 70 000 Da). Enzyme activity was dependent on exogenously supplied pyridoxal 5′-phosphate and the enzyme was stable at 4°C in the presence of the coenzyme. Optimum pH for the pure enzyme was 5.0. At this pH, TDC exhibited Michaelis–Menten kinetics ( K m 0.63 mM, V max 998 units) and was highly substrate-specific for L-tyrosine. Other amino acids and L-DOPA are not converted by the protein. Tyramine acted as a mixed non-competitive inhibitor. Significant similarities in some biochemical properties were observed with the corresponding decarboxylase enzyme of Streptococcus faecalis, the sole bacterial TDC described to date.

Purification and characterization of tyrosine decarboxylase of Lactobacillus brevis IOEB 9809 isolated from wine.

Tyrosine decarboxylase (EC 4.1.1.25) (TDC) from the wine Lactobacillus brevis IOEB 9809 was purified by a rapid procedure involving anion exchange chromatography, ultrafiltration and hydrophobic interaction chromatography. The protein comprised two subunits of identical molecular mass (approximately 70000 Da). Enzyme activity was dependent on exogenously supplied pyridoxal 5'-phosphate and the enzyme was stable at 4 degrees C in the presence of the coenzyme. Optimum pH for the pure enzyme was 5.0. At this pH, TDC exhibited Michaelis-Menten kinetics (K(m) 0.63 mM, V(max) 998 units) and was highly substrate-specific for L-tyrosine. Other amino acids and L-DOPA are not converted by the protein. Tyramine acted as a mixed non-competitive inhibitor. Significant similarities in some biochemical properties were observed with the corresponding decarboxylase enzyme of Streptococcus faecalis, the sole bacterial TDC described to date.

Purification and characterization of a highly enantioselective epoxide hydrolase from Aspergillus niger.

The epoxide hydrolase from Aspergillus niger was purified to homogeneity using a four-step procedure and p-nitrostyrene oxide (pNSO) as substrate. The enzyme was purified 246-fold with 4% activity yield. The protein is a tetramer composed of four identical subunits of molecular mass 45 kDa. Maximum activity was observed at 40 degrees C, pH 7.0, and with dimethylformamide as cosolvent to dissolve pNSO. Hydrolysis of pNSO was highly enantioselective, with an E value (i.e. enantiomeric ratio) of 40 and a high regioselectivity (97%) for the less hindered carbon atom of the epoxide. This enzyme may be a good biocatalyst for the preparation of enantiopure epoxides or diols.

A methenyl tetrahydromethanopterin cyclohydrolase and a methenyl tetrahydrofolate cyclohydrolase in Methylobacterium extorquens AM1.

Recently it was found that Methylobacterium extorquens AM1 contains both tetrahydromethanopterin (H4MPT) and tetrahydrofolate (H4F) as carriers of C1 units. In this paper we report that the aerobic methylotroph contains a methenyl H4MPT cyclohydrolase (0.9 U x mg-1 cell extract protein) and a methenyl H4F cyclohydrolase (0.23 U x mg-1). Both enzymes, which were specific for their substrates, were purified and characterized and the encoding genes identified via the N-terminal amino acid sequence. The purified methenyl H4MPT cyclohydrolase with a specific activity of 630 U x mg-1 (Vmax = 1500 U x mg-1; Km = 30 microm) was found to be composed of two identical subunits of molecular mass 33 kDa. Its sequence was approximately 40% identical to that of methenyl H4MPT cyclohydrolases from methanogenic archaea. The methenyl H4F cyclohydrolase with a specific activity of 100 U x mg-1 (Vmax = 330 U x mg-1; Km = 80 microm) was found to be composed of two identical subunits of molecular mass 22 kDa. Its sequence was not similar to that of methenyl H4MPT cyclohydrolases or to that of other methenyl H4F cyclohydrolases. Based on the specific activities in cell extract and from the growth properties of insertion mutants it is suggested that the methenyl H4MPT cyclohydrolase might have a catabolic, and the methenyl-H4F cyclohydrolase an anabolic function in the C1-unit metabolism of M. extorquens AM1.

Crotonobetaine reductase from Escherichia coli consists of two proteins

Crotonobetaine reductase from Escherichia coli is composed of two proteins (component I (CI) and component II (CII)). CI has been purified to electrophoretic homogeneity from a cell-free extract of E. coli O44 K74. The purified protein shows l(−)-carnitine dehydratase activity and its N-terminal amino acid sequence is identical to the caiB gene product from E. coli O44 K74. The relative molecular mass of CI has been determined to be 86 100. It is composed of two identical subunits with a molecular mass of 42 600. The isoelectric point of CI was found to be 4.3. CII was purified from an overexpression strain in one step by ion exchange chromatography on Fractogel EMD TMAE 650(S). The N-terminal amino acid sequence of CII shows absolute identity with the N-terminal sequence of the caiA gene product, i.e. of the postulated crotonobetaine reductase. The relative molecular mass of the protein is 164 400 and it is composed of four identical subunits of molecular mass 41 500. The isoelectric point of CII is 5.6. CII contains non-covalently bound FAD in a molar ratio of 1:1. In the crotonobetaine reductase reaction one dimer of CI associates with one tetramer of CII. A still unknown low-molecular-mass effector described for the l(−)-carnitine dehydratase is also necessary for crotonobetaine reductase activity. Monoclonal antibodies were raised against the two components of crotonobetaine reductase.

Purification and Characterization of a Polyphenol Oxidase from the Kew Cultivar of Indian Pineapple Fruit

Three isoforms of polyphenol oxidase (PPO) were purified to apparent homogeneity from the Kew cultivar of Indian pineapple fruit in a four-step procedure. The major isoenzyme, with a yield of 45%, was found to be a tetramer of identical subunits of molecular mass ≈25 kDa. An ionic strength dependent association−dissociation equilibrium was observed with pineapple PPO. Amino acid analysis of the major isoenzyme indicated the presence of a high content of glutamic acid, glycine, and serine and a low content of the sulfur-containing amino acids. The enzyme was optimally active between pH 6 and 7. The PPO did not show any cresolase activity, and the preferred substrates were diphenols. Ascorbic acid, l-cysteine, and potassium metabisulfite were found to be potent inhibitors of PPO. Keywords: Catecholase; pineapple fruit; polyphenol oxidase; PPO-isoenzyme; association−dissociation

Polythionate degradation by tetrathionate hydrolase of Thiobacillus ferrooxidans.

Cell-free extracts of Thiobacillus ferrooxidans grown with thiosulfate as energy source and prepared at high ammonium sulfate concentrations and at low pH are capable of polythionate hydrolysis. The enzyme responsible for the hydrolysis of tetrathionate (S4O2- 6) and pentathionate (S4O2- 6) was purified to homogeneity. Enzyme activity during the purification procedure was based on a continuous spectrophotometric method that detects soluble intermediates that absorb in the UV region. The end products of hydrolysis of both polythionates by the pure enzyme were thiosulfate, sulfur and sulfate. The purified enzyme has a pH optimum of around 4 and a temperature optimum of 65 �. The activity is strongly influenced by the presence of sulfate ions. The purified enzyme is a dimer with two identical subunits of molecular mass 52 kDa. During purification of tetrathionate hydrolase, fractions able to hydrolyse trithionate and tetrathionate were separated, indicating that the two substrates are hydrolysed by different enzymes.

Purification and characterisation of xylitol dehydrogenase from Neurospora crassa

The purification and characterisation of one of the key enzymes in the conversion of xylose to ethanol, xylitol dehydrogenase (XD), from Neurospora crassa is reported. This organism has the unique ability to directly convert biomass to ethanol. The enzyme was purified 81-fold by fractional ammonium sulfate precipitation, column chromatography on DEAE-cellulose, Sephacryl S-200 and β-NAD agarose. The purified enzyme had a native molecular mass of 87 kDa (gel filtration) and was composed of two identical subunits of molecular mass 43.6 kDa (SDS-PAGE). The enzyme was most active at pH 8.4 and at 28°C. It was most stable at pH 7 and at 4°C. It was activated by Mg 2+ and stabilised by Ca 2+, Mg 2+ and Mn 2+. It showed activity towards xylitol and sorbitol, the respective K ms being 28.5 and 100 mM and the K m for NAD was 0.7 mM.

L-Malate Dehydrogenase fromPseudomonas stutzeri:Purification and Characterization

L-Malate dehydrogenase (MDH) fromPseudomonas stutzeriwas purified to homogeneity by a two-step procedure comprising anion-exchange chromatography and affinity chromatography on immobilized anthraquinone α-ketocarboxyl biomimetic dye. The enzyme has molecular mass of 66,500 Da and consists of two identical subunits of molecular mass of approximately 34,000 Da. Initial velocity, product inhibition, and binding studies were consistent with an ordered Bi-Bi mechanism for the enzyme action and the formation of a ternary complex. The enzyme is susceptible to activation and inhibition by its substrates. Thermodynamic analysis and kinetic inhibition studies were performed for determining basic equilibrium and kinetic constants. Malate dehydrogenase was covalently inactivated by a dichlorotriazine dye, Vilmafix Blue A-R (VBAR). The inactivation process follows first-order kinetics, and the results from kinetic analysis suggested the formation of a noncovalent enzyme–dye complex prior to the covalent reaction, withKd84.6 μMand a maximum rate constant 0.16 min−1. The enzyme inactivation process was partially inhibited by substrates and inhibitors. Quantitatively inactivated MDH contained approximately 1 mole of dye per mole of enzyme subunit. The denatured enzyme contains 10 sulfhydryl groups per subunit, as shown after reaction with 5,5′-dithio-bis-(2-nitrobenzoic acid), of which 5 can be titrated also in the native enzyme, exhibiting time-dependent reactivity. One sulfhydryl group is located in the coenzyme binding site. This study shows that the physical and catalytic properties ofP. stutzeriMDH strongly resemble those of the mitochondrial eukariotic enzyme. This finding strengthens the existing view that, in the evolution process, the mitochondrial MDH might have appeared before the cytoplasmic.

Purification and characterization of a plasma membrane ferricyanide-utilizing NADH dehydrogenase from Ehrlich tumour cells

A ferricyanide-utilizing NADH dehydrogenase (NADH-ferricyanide oxidoreductase) from the plasma membrane of Ehrlich ascites tumour cells has been purified about 1500-fold to apparent homogeneity. The method comprises the isolation of an enriched plasma membrane fraction, solubilization with Triton X-100, ion-exchange chromatography, ammonium sulphate precipitation, Cibacron Blue chromatography and fast-protein liquid chromatography with a Superose-6 gel filtration column. The specific activity of the final pool was more than 61 units/mg protein. The pure enzyme examined by SDS/PAGE displayed only one type of subunit with an apparent molecular mass of 32.0 kDa. The molecular mass of the native protein (117.0 kDa) was estimated by gel filtration; these results suggest a protein composed of four subunits of identical molecular mass. The enzyme was stable in the pH interval between 6 and 9, with maximum activity at pH values from 7.5 to 8.5. The purified enzyme showed Michaelis-Menten kinetics for the substrates, with apparent K(m) values of 4.3 X 10(-5) M and 6.7 X 10(-5) M for NADH and ferricyanide respectively. The isolated protein was strongly inhibited by Zn2+ and the thio-specific reagents mersalyl and p-chloromercuribenzenesulphonic acid.

Occurrence of a novel L-2,4-diaminobutyrate decarboxylase activity in some species of Enterobacteriaceae, and purification and characterization of the enzymes of Enterobacter aerogenes and Serratia marcescens.

L-2,4-Diaminobutyrate decarboxylase (DABA DC) is a novel enzyme yielding 1,3-diaminopropane (DAP) from DABA, which has previously been purified from strains of the genera Vibrio and Acinetobacter. In this study, we also detected DABA DC activity in the species of Enterobacteriaceae: E. aerogenes, E. cloacae, E. agglomerans, Serratia marcescens, S. liquefaciens, Klebsiella pneumoniace, K. oxytoca and Citrobacter freundii, all of which produced DAP in sufficient amounts. Subsequently, the DABA DCs of E. aerogenes and S. marcescens were purified to homogeneity and characterized. Two separate enzymes had similar properties with respect to chromatographic behaviors, and were a dimer with subunits of identical molecular mass of about 51 kDa. The maximal activity of each enzyme was obtained at pH 8.0-8.25. Both enzymes required pyridoxal 5'-phosphate and Mg2+ for full activity, and were highly specific for L-DABA. There was immunological similarity, but not identity between these proteins, as determined by Ouchterlony double diffusion analysis with antiserum against the E. aerogenes DABA DC. They showed the same N-terminal amino acid sequence up to the 8th residue (S-K-L-N-P-I-L-A-). These enzymes were different in molecular mass, N-terminal amino acid sequence and antigenicity from DABA DCs of Acientobacter and Vibrio species.

Purification and characterization of α- d-mannosidase from Aspergillus sp.

α- d-Mannosidase from Aspergillus sp. was purified to homogeneity by preparative polyacrylamide gel electrophoresis (PAGE). The native enzyme of molecular mass 412 kDa (gel filtration) is made up of six identical subunits of molecular mass 69.2 kDa (SDS-PAGE) The enzyme is acidic (p I 4.5) and a glycoprotein with a carbohydrate content of 3.8%. The pH and temperature optima of the enzyme were in the range 6.0–6.5 and 50–55°C, respectively. At pH 6.0 the enzyme was stable for 30 min at 50°C. The K m and V max for p-nitrophenyl-α- d-mannopyranoside were 83 μM and 0.2 μmol/min per mg of the enzyme, respectively. The enzyme was strongly inhibited by 1 mM Hg 2+ and Cu 2+ and partially by 30 mM glucose and mannose. The enzyme hydrolysed Man-α-(1–3)Man at a very high rate followed by Man-α-(1–2)Man, while the rate of hydrolysis was low for Man-α-(1–6)Man. The rate of hydrolysis for high mannose oligosaccharide Man-6 was higher than for Man-9 and yeast mannan was not hydrolysed at all.

Biochemical and Molecular Characterization of Nucleoside Triphosphate Hydrolase Isozymes from the Parasitic Protozoan Toxoplasma gondii

We have previously reported the presence of a novel nucleoside triphosphate hydrolase (NTPase) in the rapidly multiplying tachyzoite form of a virulent strain (RH) of Toxoplasma gondii. On further examination, it was found that the purified enzyme was not a single enzyme but was a mixture of two isozymes termed NTPase-I and NTPase-II. The two isozymes had similar molecular masses of approximately 240-270 kDa by gel filtration and contained four identical subunits of molecular mass 66-67 kDa by SDS-polyacrylamide gel electrophoresis. Both forms of the NTPase were activated by dithiothreitol, and NTPase-I had a specific activity 4.5-fold higher than NTPase-II in hydrolysis of ATP. The primary difference between these isozymes lies in their ability to hydrolyze nucleoside triphosphate versus diphosphate substrates. While NTPase-II hydrolyzed ATP to ADP and ADP to AMP at almost the same rate, NTPase-I hydrolyzed ADP to AMP at a much slower rate (0.7% of the rate for ATP). The complete cDNAs for NTPase-I and NTPase-II were sequenced and found to encode the same size predicted open reading frame of which only 16 of 628 amino acids differed between the two isozymes. Both forms of the NTPase contained an NH2-terminal hydrophobic signal peptide, consistent with our previous findings that these enzymes are secreted into the host cell vacuole occupied by the parasite. The gene encoding NTPase-II was found in all strains of Toxoplasma, while the NTPase-I was confined only to virulent strains. Expression of this highly active ATPase (NTPase-I) may contribute to intracellular survival and virulence of T. gondii.

Purification and characterization of methionine γ-lyase from Trichomonas vaginalis

Methionine gamma-lyase (EC 4.4.1.11) was purified to homogeneity from the anaerobic protozoan parasite Trichomonas vaginalis by a series of f.p.l.c. procedures. The enzyme catalyses alpha gamma- and alpha beta-elimination reactions of a number of derivatives of methionine and cysteine. It also catalyses gamma-replacement reactions of the thiomethyl group of methionine, homocysteine and ethionine to yield the corresponding S-substituted homocysteine derivative. The enzyme is pyridoxal 5'-phosphate-dependent, has a native molecular mass of approx. 160 kDa and consists of four apparently identical subunits of molecular mass 43-45 kDa. The absorption spectrum of the enzyme is typical of those obtained for other pyridoxal 5'-phosphate-dependent enzymes, and the holoenzyme can be resolved to the apoenzyme by incubation with hydroxylamine and reconstituted by addition of the cofactor. The enzyme activity is significantly affected by carbonyl and thiol reagents, is competitively inhibited by a number of substrate analogues and is completely inactivated by the suicide inhibitor DL-propargylglycine. The T. vaginalis enzyme is similar, in terms of activity and properties, to the enzymes found in a number of species of bacteria that metabolize methionine under anaerobic conditions. It is suggested that methionine catabolism may be of particular importance to the survival of T. vaginalis under microaerophilic conditions in its host.

A novel nitrilase, arylacetonitrilase, of Alcaligenes faecalis JM3

A new type of nitrilase, arylacetonitrilase, has been purified from isovaleronitrile-induced cells of Alcaligenes faecalis JM3 in four steps. The purity of the enzyme was confirmed by SDS/polyacrylamide gel electrophoresis, ampholyte electrofocusing and double immunodiffusion in agarose. The enzyme has a molecular mass of about 275 kDa and consists of six subunits of identical molecular mass. The purified enzyme exhibits a pH optimum of 7.5 and a temperature optimum of 45 degrees C. The enzyme is specific for arylacetonitriles such as 2-thiophenacetonitrile, p-tolyacetonitrile, p-chlorobenzylcyanide, p-fluorobenzylcyanide and 3-pyridylacetonitrile. The enzyme stoichiometrically catalyzes the hydrolysis of arylacetonitrile to arylacetic acid and ammonia, no formation of amide occurring. However, the enzyme does not attack nitrile groups attached to aromatic and heteroaromatic rings, which are hydrolyzed preferably by the nitrilases known previously. The enzyme requires thiol compounds such as dithiothreitol and 2-mercaptoethanol to exhibit its maximum activity.