Active enzyme gel chromatography: II. Computer simulations

The behavior of an enzyme undergoing reaction while on a gel chromatography column has been studied by computer simulation using the steady state assumption for a system with a single enzyme—substrate complex. The profiles of the enzyme—substrate complex, product, and substrate were examined varying the parameters of k_cat, flow rate, partition coefficient, dispersion coefficient, and time. These investigations confirm that much information about both the active enzyme and the product may be obtained by examining the product profile alone, varying the power of applying scanning gel chromatography to active enzyme systems.     

Interaction of Mg2+ with human liver aldehyde dehydrogenase. I. Species difference in the mitochondrial isozyme

The dehydrogenase activity of the mitochondrial isozyme (E2) of human liver aldehyde dehydrogenase was stimulated about 2-fold by the presence of low concentrations (about 120-140 microM) of Mg2+ in the assay at pH 7.0 using propionaldehyde as substrate. The stimulation was totally reversible by treatment with EDTA. Maximum stimulation was dependent on the concentration of NAD+ used in the assay; an increase in Km value of NAD+ was observed to parallel the increase in maximal velocity with increasing Mg2+ concentration, indicating that alterations in the catalytic properties of the E2 isozyme occur in the presence of Mg2+. The presteady state burst of NADH product was observed to decrease in the presence of Mg2+, suggesting that the rate-limiting step of the dehydrogenase reaction is altered by Mg2+. No evidence for Mg2+-induced alterations in the molecular weight properties of the E2 isozyme was observed using gel filtration column chromatography and fluorescence polarization techniques. In addition, no alterations in the inactivating properties of iodoacetamide or disulfiram were produced by Mg2+. These results suggest that the mechanism by which human mitochondrial aldehyde dehydrogenase (E2) is stimulated by Mg2+ is different from that of the horse enzyme, representing a significant species difference.     

Active-site-specific zinc-depleted and reconstituted cobalt(II) human-liver alcohol dehydrogenase. Preparation, characterization and complexation with NADH and trans-4-(N,N-dimethylamino)-cinnamaldehyde

The active-site zinc atom of the beta 1 beta 1 isozyme of class I alcohol dehydrogenase (EC 1.1.1.1) from human liver was specifically removed by the chelating agent dipicolinic acid. From beta 1 gamma 1 and gamma 1 gamma 1 isozyme the active-site zinc is extracted much more slowly than from beta 1 beta 1 isozyme. Only partially active-site metal-depleted enzyme species were obtained from these isozymes. The active-site-specific reconstituted cobalt(II) derivative of the beta 1 beta 1 isozyme shows spectroscopic properties comparable to those of the active-site-specific reconstituted cobalt(II) horse liver alcohol dehydrogenase. The coenzyme-induced conformational change of the protein leads to a red shift of the d-d band from 648 nm to 673 nm. The chromophoric substrate trans-4-(N,N-dimethylamino)-cinnamaldehyde forms ternary complexes with NADH and the different isozymes, in close analogy to horse liver alcohol dehydrogenase. The differences in the active sites between beta 1 and gamma 1 subunits (threonine-48 instead of serine-48) or between zinc and cobalt(II) are reflected in the visible absorption spectra of the metal-bound chromophoric substrate.     

Metabolism of n-propylamine, isopropylamine, and 1,3-propane diamine by Mycobacterium convolutum.

Mycobacterium convolutum strain NPA-1 can utilize n-propylamine (NPA), isopropylamine (IPA), and 1,3-propane diamine (PD) as sole source of carbon, nitrogen, and energy. Enzyme assays, fatty acid profiles, and 14CO2 incorporation experiments indicate that NPA is deaminated to propionate and further metabolized via the methylmalonyl succinate pathway, and IPA and PD were metabolized (after deamination) through a C2 + C1 cleavage. An inducible amine dehydrogenase was present in cell extracts after growth on the three amines. Polyacrylamide gel electrophoresis of cell extracts from NPA- and IPA-grown cells yielded one major band of amine dehydrogenase activity. When extracts of NPA-grown cells were assayed with NPA, IPA, or PD as substrate, the relative position of the major band on gel electrophoresis was equivalent. Similar results were obtained with extracts prepared from IPA-grown cells. Sephadex G-100 chromatography also indicated one major peak of activity. This suggests that one enzyme of broad specificity is involved in deamination of IPA, NPA, and PD. IPA-grown cells utilized NPA readily, whereas NPA-grown cells could not utilize IPA without lag. Since amine dehydrogenase activity was present in extracts of cells after growth on either substrate, this lag was probably due to the inability to transport IPA without an induction period. The molecular weight of the amine dehydrogenase was approximately 38,500 as determined by gel filtration.     

Purification and properties of the NAD+-xylitol-dehydrogenase from the yeast Pichia stipitis

Cell-free extracts of the xylose fermenting yeast Pichia stipitis exhibited xylitol dehydrogenase activity with NAD⁺ and NADP⁺. During the purification step on DEAE-sephadex A-50 a NAD⁺-dependent xylitol dehydrogenase could be separated from a NADP⁺-dependent. The NAD⁺-xylitol dehydrogenase was further purified to electrophoretic homogeneity via gel and affinity chromatography. The purified enzyme was most active at pH 9 and 35°C. Its molecular weight was determined to be 63,000 dalton by Sephadex G-200 column chromatography, and that of its subunit was 32,000 dalton by sodium dodecyl sulphate polyacrylamide gel electrophoresis. From the results of substrate specificity, the enzyme should be named l-iditol:NAD⁺-5-oxidoreductase (EC 1.1.1.14, sorbitol dehydrogenase).     

3alpha-, 7alpha- and 12alpha-hydroxysteroid dehydrogenase activities from Clostridium perfringens.

25 strains of Clostridium perfringens were screened for hydroxysteroid dehydrogenase activity; 19 contained NADP-dependent 3alpha-hydroxysteroid dehydrogenase and eight contained NAD-dependent 12alpha-hydroxysteroid dehydrogenase active against conjugated and unconjugated bile salts. All strains containing 12alpha-hydroxysteroid dehydrogenase also contained 3alpha-hydroxysteroid dehydrogenase although 12alpha-hydroxysteroid dehydrogenase was invariably in lesser quantity than the 3alpha-hydroxysteroid dehydrogenase. In addition, 7alpha-hydroxysteroid dehydrogenase activity was evident only when 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholanoate was substrate but notably absent when 3alpha, 7alpha-dihydroxy-5beta-cholanoate was substrate. The oxidation product 12alpha-hydroxy-3, 7-diketo-5beta-cholanoate is rapidly further degraded to an unknown compound devoid of either 3alpha- or 7alpha-OH groups. Group specificity of these enzymes was confirmed by thin-layer chromatography studies of the oxidation products. These enzyme systems appear to be constitutive rather than inducible. In contrast to C. perfringens. Clostridium paraputrificum (five strains tested) contained no measurable hydroxysteroid dehydrogenase activity. pH studies of the C. perfringens enzymes revealed a sharp pH optimum at pH 11.3 and 10.5 for the 3alpha-OH- and 12alpha-OH-oriented activities, respectively. Kinetic studies gave Km estimates of approx. 5 X 10(-5) and 8 X 10(-4) M with 3alpha, 7a-dihydroxy-5beta-cholanoate and 3alpha, 12alpha-dihydroxy-5beta-cholanoate as substrates for two respective enzymes. 3alpha-hydroxysteroid dehydrogenase was active against 3alpha-OH-containing steroids such as androsterone regardless of the sterochemistry of the 5H (Both A/B cis and A/B trans steroides were substrates). There was no activity against 3beta-OH-containing steroids. The 3alpha- and 12alpha-hydroxysteroid dehydrogenase activities, although differing in cofactor requirements cannot be distinguished by their appearance in the growth curve, their mobility on disc gel electrophoresis, elution volume on passage through Sephadex G-200 or heat inactivation studies.     

Bacterial cis-dihydrodiol dehydrogenases: comparison of physicochemical and immunological protperties.

Cells of Pseudomonas putida NP, Pseudomonas species (NCIB 9816), and a Nocardia species, after growth on naphthalene as sole source of carbon and energy, contain a nicotinamide adenine dinucleotide (NAD+)-dependent enzyme that oxidizes cis-dihydrodiols of mono- and polycyclic aromatic compounds. Similarly, cells of a strain of P. putida biotype A, when grown either on toluene or benzene vapors, were found to contain a dehydrogenase that oxidized dihydrodiols of aromatic hydrocarbons with cis stereochemistry and required NAD+ as an electron acceptor. In all these cases, no enzymatic activity was detected when trans-naphthalene dihydrodiol was used as a substrate. Purified cis-naphthalene dihydrodiol dehydrogenase was injected into rabbits to obtain antibodies. Physiocochemical and immunological properties of cis-dihydrodiol:NAD+ oxidoreductases from four different organisms were examined. Kinetic analysis showed that, in all the cases, enzymes exhibited higher affinity for cis-dihydrodiols than for NAD+ and had pH optima between 8.8 and 9.0. except in the case of the enzyme from Nocarida sp., which showed maximum activity at pH 8.4. Molecular-weight determination of the dehydrogenases from the four different organisms by gel filtration on a Sephadex G-200 column gave values ranging from 92,000 for the enzyme from Nocardia sp. to 160,000 for that from P. putida biotype A. All the dehydrogenases, except the one from Nocardia sp., exhibited immunological cross-reaction with the antibodies prepared against the enzyme purified from P. putida NP.     

Radiation-induced inactivation of dihydroorotate dehydrogenase in dilute aqueous solution.

The inactivation of dihydroorotate dehydrogenase by gamma irradiation in dilute aqueous solution has been investigated. The activity of the enzyme decreased exponentially as a function of the absorbed dose under aerated and nitrous oxide-saturated conditions. The contributions of the individual radical species derived from water radiolysis were estimated from the inactivation results observed under aerated, argon-saturated, and nitrous oxide-saturated conditions. The hydrogen atom and hydroxyl radical were found to be important in enzyme inactivation. The effect of selected inorganic radical anions such as Br.2-, I.2-, and (SCN).2- on the enzyme activity was also studied, and the results implicate the possible involvement of cysteine and tyrosine residues in the catalytic activity of dihydroorotate dehydrogenase. Changes in the kinetic parameters (Michaelis-Menten constant, Km, and maximal velocity, Vmax) due to irradiation under the conditions investigated suggest that radiation-induced inactivation is due to modification of the substrate binding sites and that of the active site residues in the enzyme. Evidence for the reduction of iron-sulfur centers in the enzyme during the inactivation process has been put forward from the difference spectrum of the irradiated dihydroorotate dehydrogenase. It has also been shown by electrophoretic studies that radiation-induced inactivation was not due to any fragmentation of the protein structure or the formation of any intermolecular crosslinking.     

Subunit interactions in liver alcohol dehydrogenase: A partial alkylation study.

The transient kinetics of aldehyde reduction by NADH catalyzed by liver alcohol dehydrogenase consist of two kinetic processes. This biphasic rate behavior is consistent with a model in which one of the two identical subunits in the enzyme is inactive during the reaction at the adjacent protomer. Alternatively, enzyme heterogeneity could result in such biphasic behavior. We have prepared liver alcohol dehydrogenase containing a single major isozyme; and the transient kinetics of this purified enzyme are biphasic.Addition of two [¹⁴C]carboxymethyl groups per dimer to the two “reactive” sulfhydryl groups (Cys46) yields enzyme which is catalytically inactive toward alcohol oxidation. Alkylated enzyme, as initially isolated by gel filtration chromatography at pH 7·5, forms an NAD⁺-pyrazole complex. However, the ability to bind NAD⁺-pyrazole is rapidly lost in pH 8·75 buffer; therefore, our alkylated preparations, as isolated by chromatography at pH 8·75, are inactive toward NAD⁺-pyrazole complex formation. We have prepared partially inactivated enzyme by allowing iodoacetic acid to react with liver alcohol dehydrogenase until 50% of the NAD⁺-pyrazole binding capacity remains; under these reaction conditions one [¹⁴C]carboxymethyl group is added per dimer. This partially alkylated enzyme preparation is isolated by gel filtration and has been aged sufficiently to lose NAD⁺-pyrazole binding ability at alkylated subunits. When solutions of native liver alcohol dehydrogenase and partially alkylated liver alcohol dehydrogenase containing the same number of unmodified active sites are allowed to react with substrate under single turnover conditions, partially alkylated enzyme is only half as reactive as native enzyme. This indicates that some molecular species in partially alkylated liver alcohol dehydrogenase that react with pyrazole and NAD⁺ during the active site titration do not react with substrate. These data are consistent with a model in which a subunit adjacent to an alkylated protomer in the dimeric enzyme is inactive toward substrate. In addition, NAD⁺-pyrazole binding at the protomers adjacent to alkylated subunits is slowly lost so that 75% of the enzyme-NAD⁺-pyrazole binding capacity is lost in 50% alkylated enzyme. These data supply strong evidence for subunit interactions in liver alcohol dehydrogenase.Binding experiments performed on partially alkylated liver alcohol dehydrogenase indicate that coenzyme binding is normal at a subunit adjacent to an alkylated protomer even though active ternary complexes cannot be formed. One hypothesis consistent with these results is the unavailability of zinc for substrate binding at the active site in subunits adjacent to alkylated protomers in monoalkylated dimer.     

Kinetic properties of enzymes in particular of yeast alcohol dehydrogenase following their adsorption on polyaminomethylstyrene]

The adsorption of 8 enzymes to polyaminomethylstyrene was studied. While lactate dehydrogenase, alkaline phosphatase and glucose-6-phosphate dehydrogenase exhibit a relatively low affinity to the carrier, alcohol dehydrogenase, glutamate dehydrogenase and urease were found to form stabile complexes with the polymer that are enzymatically active. Adsorbed urease and beta-hydroxybutyrate dehydrogenase, are still active after several weeks; the other preparations lose their activity soon. It can be shown by the example of yeast alcohol dehydrogenase that the activity loss following adsorption is caused possibly by a process of reorientation of already bound enzyme molecules or by the increasing enzyme coverage of the carrier, with the active centres becoming more and more inaccessible for the substrate. During the substrate conversion catalysed by the alcohol dehydrogenase-polyaminomethylstyrene complex, a small amount of the enzyme is again detached from the carrier. The activity rises to a certain extent in the supernatant but drops to zero again. The stability of the adsorbed urease is distinctly increased compared with the dissolved enzyme. For the pH optimum and the KM value there are no differences between the two preparations. Continuous application of polyaminomethylstyrene-bound beta-hydroxybutyrate dehydrogenase and urease, respectively, in a column shows that both preparations have unchanged enzymatic activities even after running times of 5 and 24 days, respectively.     

Crystallization and Properties of Amine Dehydrogenase from Pseudomonas sp.

An amine dehydrogenase was purified and crystallized from the cell free extract of a Pseudomonas sp., isolated from soil by means of the enrichment technique. The crystalline enzyme gave a single band on polyacrylamide gel electrophoresis and the molecular weight of the enzyme was estimated to be 100,000 by gel filtration on a Sephadex column. Upon SDS-gel electrophoresis, the enzyme was dissociated into two nonidentical subunits having molecular weights of 60,000 (dehydrogenase) and 39,000 (cytochrome c). The absorption spectrum of the enzyme showed absorption maxima at 550 nm, 524 nm, 411 nm and 280 nm, and a broad shoulder at around 350 nm, indicating that the enzyme was purified as a dehydrogenase-cytochrome c complex. The prosthetic group of the dehydrogenase was identified as covalently bound pyrroloquinoline quinone. The enzyme showed a broad substrate specificity toward various amines including aliphatic monoamines, aliphatic diamines, aromatic amines and polyamines.     

Comparative characterization of human and rat liver glycogen synthase.

Glycogen synthase from human liver was studied, and its properties were compared with those of rat liver glycogen synthase. The rat and human liver glycogen synthases were similar in their pH profile, in their kinetic constants for the substrate UDP-glucose and the activator glucose 6-phosphate, and in their elution profiles from Q-Sepharose. The apparent molecular weight of the human synthase subunit was 80,000-85,000 by gel electrophoresis, which is similar to that of the rat enzyme. In addition, antibodies to rat liver glycogen synthase recognized human liver glycogen synthase, indicating that the enzymes of these two species share antigenic determinants. However, there were significant differences between the two enzymes. In particular, the total activity of the human enzyme was higher than that of the rat. The human enzyme, purified to near homogeneity, had a specific activity of 40 U/mg protein compared with 20 U/mg protein for the rat enzyme. The active forms of the rat enzyme had greater thermal stability than those of the human enzyme, but the human enzyme was more stable on storage in various buffers. Last, amino acid analysis indicated differences between the enzymes of the two species. Since glycogen synthase is an important enzyme in liver glycogen synthesis, the characterization of this enzyme in the human will help provide insight regarding human liver glycogen synthesis.     

3 beta, 17 beta-hydroxysteroid dehydrogenase of Pseudomonas testosteroni. Kinetic evidence for the bifunctional activity at a common catalytic site

3 beta, 17 beta-Hydroxysteroid dehydrogenase (3 beta 17 beta HSDH) is an NAD-dependent dehydrogenase which has a double specificity for the 3- and 17-positions on the steroid skeleton. When dehydroepiandrosterone (DHEA) is used as steroid substrate, and the assay coupled with ketosteroid-isomerase, the two reactions occur alternately and each reaction on the 3-position produces a chromophoric molecule. These two reactions can follow one another without dissociation of the coenzyme from the enzyme binding site. This is confirmed by competition experiments with another dehydrogenase.     

Purification and characterization of formaldehyde dehydrogenase from rat liver cytosol.

Formaldehyde dehydrogenase was purified to electrophoretic and column chromatographic homogeneity from rat liver cytosolic fraction by a procedure which includes ammonium sulfate precipitation, DEAE-cellulose-, hydroxyapatite-, Mono Q-chromatography, and gel filtration. Its molecular mass was estimated to be 41 kDa by gel filtration and SDS-PAGE, suggesting that it is a monomer. It utilized neither methylglyoxal nor aldehydes except formaldehyde as a substrate. It has been reported that liver class III alcohol dehydrogenase and formaldehyde dehydrogenase are the same enzyme and oxidize formaldehyde and long chain primary alcohols. However, the enzyme examined here did not use n-octanoi as a substrate. The Km values for formaldehyde and NAD+ were 5.09 and 2.34 microM at 25 degrees C, respectively. The amino acid sequences of 10 peptides obtained from the purified enzyme after digestion with either V8 protease or lysyl endopeptidase were determined. From these results, the enzyme was proved to be different from the previously described mammalian formaldehyde dehydrogenase and is the first true formaldehyde dehydrogenase to be isolated from a mammalian source.     

Purification and characterization of S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase from Pseudomonas denitrificans.

S-Adenosyl-L-methionine:uroporphyrinogen III methyltransferase (SUMT), the enzyme of the cobalamin biosynthetic pathway which catalyzes C methylation of uroporphyrinogen III, was purified about 150-fold to homogeneity from extracts of a recombinant strain of Pseudomonas denitrificans derived from a cobalamin-overproducing strain by ammonium sulfate fractionation, anion-exchange chromatography, and hydroxyapatite chromatography. The purified protein has an isoelectric point of 6.4 and molecular weights of 56,500 as estimated by gel filtration and 30,000 as estimated by gel electrophoresis under denaturing conditions, suggesting that the active enzyme is a homodimer. It does not contain a chromophoric prosthetic group and does not seem to require metal ions or cofactors for activity. SUMT catalyzes the two successive C-2 and C-7 methylation reactions involved in the conversion of uroporphyrinogen III to precorrin-2 via the intermediate formation of precorrin-1. In vitro studies suggest that the intermediate monomethylated product (precorrin-1) is released from the protein and then added back to the enzyme for the second C-methylation reaction. The pH optimum was 7.7, the Km values for S-adenosyl-L-methionine and uroporphyrinogen III were 6.3 and 1.0 microM, respectively, and the turnover number was 38 h-1. The enzyme activity was shown to be completely insensitive to feedback inhibition by cobalamin and corrinoid intermediates tested at physiological concentration. At uroporphyrinogen III concentrations above 2 microM, SUMT exhibited a substrate inhibition phenomenon. It is suggested that this property might play a regulatory role in cobalamin biosynthesis in the cobalamin-overproducing strain studied.     

Kinetics and stability of immobilized chicken liver xanthine dehydrogenase.

Xanthine dehydrogenase (EC 1.2.1.37) was isolated from chicken livers and immobilized by adsorption to a Sepharose derivative, prepared by reaction of n-octylamine with CNBr-activated Sepharose 4B. Using a crude preparation of enzyme for immobilization it was observed that relatively more activity was adsorbed than protein, but the yield of immobilized activity increased as a purer enzyme preparation was used. As more activity and protein were bound, relatively less immobilized activity was recovered. This effect was probably due to blocking of active xanthine dehydrogenase by protein impurities. The kinetics of free and immobilized xanthine dehydrogenase were studied in the pH range 7.5-9.1. The Km and V values estimated for free xanthine dehydrogenase increase as the pH increase; the K'm and V values for the immobilized enzyme go through a minimum at pH 8.1. By varying the amount of enzyme activity bound per unit volume of gel, it was shown that K'm is larger than Km are result of substrate diffusion limitation in the pores of the support material. Both free and immobilized xanthine dehydrogenase showed substrate activation at low concentrations (up to 2 microM xanthine). Immobilized xanthine dehydrogenase was more stable than the free enzyme during storage in the temperature range of 4-50 degrees C. The operational stability of immobilized xanthine dehydrogenase at 30 degrees C was two orders of magnitude smaller than the storage stability, t 1/2 was 9 and 800 hr, respectively. The operational stability was, however, better than than of immobilized milk xanthine oxidase (t 1/2 = 1 hr). In addition, the amount of product formed per unit initial activity in one half-life, was higher for immobilized xanthine dehydrogenase than for immobilized xanthine oxidase. Unless immobilized milk xanthine oxidase can be considerable stabilized, immobilized chicken liver xanthine dehydrogenase is more promising for application in organic synthesis.     

Purification,properties, and kinetic mechanism of coenzyme A-linked aldehyde dehydrogenase from Clostridium kluyveri

Coenzyme A-linked aldehyde dehydrogenase from Clostridium kluyveri was purified from the soluble fraction of crude extracts and its physical and kinetic properties were studied. The enzyme was purified approximately 90-fold over crude extracts to a specific activity of 50 units/mg protein and was estimated to be 40% pure by polyacrylamide gel electrophoresis. From active enzyme centrifugation studies, aldehyde dehydrogenase was found to have a sedimentation coefficient of s_{20, w} = 7.4. The Stokes radius of the enzyme was determined by gel filtration and found to be 9.5 nm in the presence of substrates and 11.0 nm in the absence of substrates. Using the values found for the sedimentation coefficient and the Stokes radius, the molecular weight of the enzyme in the presence of substrates was calculated to be 290,000 and the frictional ratio, 2.2. Aldehyde dehydrogenase can utilize thiols other than CoA as acetyl acceptors. A number of methods were employed in order to exclude the possibility that these thiols act merely by recycling nonenzymatically trace amounts of CoA that might be in the enzyme preparation. From steady-state kinetic measurements, a ping pong mechanism was proposed in which NAD⁺ binds to free enzyme, acetaldehyde binds next, and NADH is released before CoA binds and acetyl-CoA released. At K_m levels of other substrates, substrate inhibition by CoA was observed. The nature of the substrate inhibition is discussed.     

Histochemical modification of the active site of succinate dehydrogenase with N-acetylimidazole

The kinetics of acetylation of mitochondrial succinate dehydrogenase [EC 1.3.99.1] in the two fibre types (A and C) of rat gastrocnemius with N-acetylimidazole was studied by a newly modified histochemical technique. Acetylimidazole partially inactivated the enzyme, but subsequent deacetylation with hydroxylamine restored the enzyme activity completely. Inactivation of the enzyme by acetylimidazole was prevented by malonate, which is a competitive inhibitor of the enzyme. The value of the inhibition constant (Ki = 34 microM) for malonate, obtained from the dependence of the pseudo-first order rate constant of acetylation of the enzyme with acetylimidazole on the malonate concentration, was in good agreement with the Ki value (33 microM) obtained by a different method, the dependence of the initial velocity of succinate oxidation by the dehydrogenase on the substrate concentration in the presence of malonate. These findings suggest that a tyrosyl residue is located in the malonate binding site (the active site) of succinate dehydrogenase in the gastrocnemius and plays a role in substrate binding, but is not a catalytic group.     

Studies on a 2,3-diaminopropionate: ammonia-lyase from a Pseudomonad.

2,3-Diaminopropionate:ammonia-lyase, an induced enzyme in a Pseudomonas isolate, has been purified 40-fold and found to be homogeneous by disc gel electrophoresis and by ultracentrifugation. Some of its properties have been studied. The optimum pH and temperature for activity are 8 and 40 degrees C, respectively. The enzyme shows a high degree of substrate specificity, acting only on 2,3-diaminopropionate; the D-isomer is only one-eighth as effective as the L-form. L-Homoserine and DL-cystathionine are not substrates, and 3-cyanolalanine does not inhibit its activity. It is a pyridoxal phosphate enzyme which requires free enzyme sulphhydryls for activity. The Km values for L-2,3-diaminopropionate and pyridoxal phosphate are 1mM and 25 muM, respectively. The molecular weight of the enzyme is about 80 000 as determined by gel filtration. On treatment with 0.5M urea or guanidine by hydrochloride, the enzyme dissociates into inactive subunits with an approximate molecular weight of 45 000. One mole of the active enzyme binds one mole of pyridoxal phosphate. The bacterial enzyme seems to be quite different in many of its properties from the rat liver enzyme which also exhibits the substrate specificity of cystathionine gamma-lyase.     

Reactivity and specificity of alpha-dicarbonyl compounds towards essential aminoacyl residues of D-beta-hydroxybutyrate dehydrogenase from the inner mitochondrial membrane

The effects of a alpha-dicarbonyl chromophoric reagent: 4-hydroxy-3-nitrophenylglyoxal on the D-beta-hydroxybutyrate dehydrogenase have been compared to those of phenylglyoxal, a specific arginyl reagent in proteins. Both reagents inactivate irreversibly the enzyme. Kinetic experiments show that only one molecule of these reagents per molecule of enzyme is sufficient to inactivate the enzyme. The second order inactivation rate constant is more than 500 times higher with the chromophoric reagent than with phenylglyoxal. A pseudosubstrate (methylmalonate) in presence of coenzyme (NAD) strongly protects enzyme against inactivation by both reagents. Coenzyme alone has no effect on inactivation by phenylglyoxal while it protects whether inhibitor is the chromophoric reagent or N-ethylmaleimide: a thiol specific reagent. These results indicate: 1. That one arginyl residue is essential for D-beta-hydroxybutyrate dehydrogenase activity (experiments with phenylglyoxal). 2. That the presence of a nitro group on position 3 and a hydroxyl-group on position 4 strongly increase the reactivity of the alpha-dicarbonyl groups, but the specificity of the chemical reaction with arginyl residues seems to be lost for the benefit of cysteyl residues.