固定化谷氨酸棒杆菌T6—13细胞的酶学性质研究

比较研究了固定化谷氨酸棒杆菌细胞和自然细胞的谷氨酸脱氢酶、异拧檬酸脱氢酶,葡萄糖-6-磷酸脱氢酶的一些性质。最适pH、温度对二者酶促反应速度的影响基本相似;pH、热稳定性固定化细胞高于自然细胞;底物表观米氏常数谷氨酸脱氢酶,异柠檬酸脱氢酶有所增大,而葡萄糖-6-磷酸脱氢酶则有所下降;辅酶表观米氏常数均有所增大。这些是影响固定化细胞应用的主要因素。     

The significance of abrupt transitions in Lineweaver–Burk plots with particular reference to glutamate dehydrogenase. Negative and positive co-operativity in catalytic rate constants

1. Lineweaver-Burk plots for glutamate dehydrogenase, glucose 6-phosphate dehydrogenase and several other enzymes show one or more abrupt transitions between apparently linear sections. These transitions correspond to abrupt increases in the apparent K(m) and V(max.) with increasing concentration of the varied substrate. 2. The generalized reciprocal initial-rate equation for a multi-site enzyme requires several restrictions to be put on it in order to generate such plots. These mathematical conditions are explored. 3. It is shown that the effective omission of a term in the denominator of the reciprocal initial-rate equation represents a minimal requirement for generation of abrupt transitions. This corresponds in physical terms to negative co-operativity followed by positive co-operativity affecting the catalytic rate constant for the reaction. 4. Previous models for glutamate dehydrogenase cannot adequately account for the results. On the other hand, the model based on both negative and positive co-operativity gives a good fit to the experimental points. 5. The conclusions are discussed in relation to current knowledge of the structure and mechanism of glutamate dehydrogenase.     

Structural organization of glutamate dehydrogenase hexamer. 1. Immobilization on sepharose as a model for analysis of structural-functional characteristics of the enzyme

Bovine liver glutamate dehydrogenase (L-glutamate-NAD(P)-oxidoreductase, EC 1.4.1.3) and its radioactive phosphopyridoxyl derivative were covalently immobilized on Sepharose CL-4B with different degrees of cyanogen bromide activation. The catalytical and regulatory properties of the immobilized samples of the enzymes were studied. It was shown that the enzymes were immobilized through a single subunit of hexamer when sepharose was activated by small amounts of cyanogen bromide (less than 5 mg per 1 ml of gel). In this case, the immobilization did not alter the catalytical and regulatory properties of glutamate dehydrogenase. The immobilized radioactive phosphopyridoxyl derivative of glutamate dehydrogenase completely imitated the immobilized native enzyme and can be used as a convenient model for structural and functional investigation of catalytically active hexamer of glutamate dehydrogenase.     

具有谷氨酸脱羧酶的大肠杆菌固定化细胞

本文研究了用海藻酸钙包埋法制备含谷氨酸脱羧酶固定化细胞的方法以及研究了制备的条件和影响其制备的因素。该法具有包埋细胞活力回收高,方法简便等优点。比较研究了固定化细胞和自然细胞谷氨酸脱羧酶的一些生物化学性质。其中固定化细胞最适pH和pH稳定性增加,最适温度及热稳定性下降;表观米氏常数增大;二价金属离子Zn~(++)、Cu~(++)、Mg~(++)、Fe~(++),Sr~(++)程度不同的抑制酶活性,Ca~(++)激活固定化细胞酶活性,EDTA无抑制作用。对固定化细胞和自然细胞酶活力活化的研究中发现这两种细胞经蒸馏水保温处理后酶活性都上升,且自然细胞酶活的上升较固定化细胞大;而用底物溶液处理后,自然细胞无变化,固定化细胞酶活下降。     

Affinity labeling of bovine liver glutamate dehydrogenase with 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-diphosphate and 5'-triphosphate

Bovine liver glutamate dehydrogenase reacts with 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-diphosphate (8-BDB-TA-5'-DP) and 5'-triphosphate (8-BDB-TA-5'-TP) to yield enzyme with about 1 mol of reagent incorporated/mol of enzyme subunit. The modified enzyme is catalytically active but has decreased sensitivity to inhibition by GTP, reduced extent of activation by ADP, and diminished inhibition by high concentrations of NADH. Since modified enzyme, like native glutamate dehydrogenase, reversibly binds more than 1 mol each of ADP and GTP, it is unlikely that 8-BDB-TA-5'-TP reacts directly within either the ADP or GTP regulatory sites. The rate constant for reaction of enzyme exhibits a nonlinear dependence on reagent concentration with KD = 89 microM for 8-BDB-TA-5'-TP and 240 microM for 8-BDB-TA-5'-DP. The ligands ADP and GTP alone and NADH alone produce only small decreases in the rate constant for the reaction of enzyme with 8-BDB-TA-5'-TP, but the combined addition of 5 mM NADH + 200 microM GTP reduces the reaction rate constant more than 10-fold and the reagent incorporation to about 0.1 mol/mol of enzyme subunit. These results suggest that 8-BDB-TA-5'-TP reacts as a nucleotide affinity label in the region of the GTP-dependent NADH regulatory site of bovine liver glutamate dehydrogenase.     

Characterization of specific interactions of coenzymes, regulatory nucleotides and cibacron blue with nucleotide binding domains of enzymes by analytical affinity chromatography

The dissociation constant for the complex of rhodanese and Cibacron Blue, determined by analytical affinity chromatography using rhodanese immobilized on controlled-pore glass (CPG) beads (200 nm pore diameter) and aminohexyl-Cibacron Blue, was 44 microM which agreed well with the kinetic inhibition constant, suggesting that the dye binds at or near the active site of this enzyme. Formation of a binary complex of the dye and lactate dehydrogenase (LDH) was also characterized by direct chromatography of LDH on CPG/immobilized Cibacron Blue (KD = 0.29 microM). The binary complex formed between LDH and NADH was characterized by analytical affinity chromatography using both CPG/immobilized LDH and immobilized Cibacron Blue. Since the dye competes with NADH in binding to the active site of LDH, competitive elution chromatography using the immobilized dye allows determination of the dissociation constant of the soluble LDH.NADH complex. Agreement between the dissociation constants determined by direct chromatography of NADH on immobilized LDH (KD = 1.4 microM) and that determined for the soluble complex (KD = 2.4 microM) indicates that immobilization of LDH did not affect the interaction. Formation of various binary, ternary and quaternary complexes of bovine liver glutamate dehydrogenase (GDH) with glutamate, NADPH, NADH, and ADP was also investigated using immobilized GDH. This approach allows characterization of the enzyme/ligand interactions without the complicating effect of enzyme self-association. The affinity for NADPH is considerably greater in the ternary complex (including glutamate) as compared to the binary complex (0.38 microM vs 22 microM); however, occupancy of the regulatory site by ADP greatly reduces the affinity in both complexes (6.4 microM and 43 microM, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of temperature on the stability and activity of crystalline ox liver nuclear and mitochondrial glutamate dehydrogenases

A study on the response of the stability and activity of crystalline ox liver nuclear and mitochondrial glutamate dehydrogenases to temperature variations has been carried out. The thermodynamic properties of the heat inactivation process and of the reaction with the substrates glutamate and α-ketoglutarate have been investigated. The heat inactivation of nuclear glutamate dehydrogenase proceeds at a faster rate than that of the mitochondrial enzyme in the temperature range 40–51 °C; the enthalpy of activation of the inactivation process is higher and the entropy is almost double, compared to the values of mitochondrial glutamate dehydrogenase. The effect of temperature on the maximal velocity shows that, with both glutamate and α-ketoglutarate, the enthalpy of activation with nuclear glutamate dehydrogenase is double and the decrease in entropy almost half of the values of the mitochondrial enzyme. The variation of the apparent K_m with temperature shows a decrease of the affinity of both enzymes for glutamate, with no major difference in the thermodynamic properties of the reaction. With α-ketoglutarate, on the other hand, the affinity of nuclear glutamate dehydrogenase decreased, whereas that of the mitochondrial enzyme increased with temperature. The process is therefore exothermic with the former enzyme, endothermic with the latter; furthermore, it occurs with a decrease in enthropy with nuclear glutamate dehydrogenase, but with a large increase with the mitochondrial enzyme. The studies on the effect of temperature on the activity were carried out in the range 20–44 °C.     

Interaction of proteins with Triton X-100-substituted sepharose 4B

Interaction of a number of arbitrarily chosen proteins with Triton X-100-substituted Sepharose 4B has been investigated. Of the proteins examined, bovine serum albumin, hemoglobin, glutamate dehydrogenase, and pepsin were found immobilized on the adsorbent. Binding of these proteins occurred irrespective of pH and NaCl concentration. Cytochrome c, used as a model protein, was totally immobilized only at low pH. Adsorption of glutamate dehydrogenase and pepsin took place with retention of their catalytic activities. Moreover, glutamate dehydrogenase used as a model allosteric enzyme, was found to retain its native properties upon binding to the adsorbent in the forms of suspension or column. Results are discussed in terms of specific interactions involving the hydrophobic region of Triton X-100 and the apolar patches or crevices present on the surface of protein molecules. Possible potential of the matrix as a method for preparation of biologically active immobilized proteins and its application in continuous operations are also discussed.     

Effect of pH and ionic strength on the catalytic and allosteric properties of native and chemically modified ox liver mitochondrial glutamate dehydrogenase.

Inorganic phosphate, a strong activator of glutamate dehydrogenase at pH 8.0–9.0, is an inhibitor at pH 6.0–7.6. The extent of inhibition increases with the decrease of pH. The same effect is shown by other electrolytes, including Tris-hydroxymethyl-aminomethane and NaCl.The combined effect of pH and ionic strength also alters the allosteric characteristics of the enzyme. Lowering the pH minimizes the activation by high concentrations of NAD; phosphate partially restores this activation. The allosteric activation by ADP disappears at pH around neutrality; in the pH range 6.0–7.0, ADP becomes a strong inhibitor, the inhibition being enhanced by the addition of ionic compounds. Similarly, the extent of allosteric inhibition by guanosine 5′-triphosphate (pyro) (GTP), which is maximal at pH 9.0, decreases at lower pH values and a slight activation is observed in the presence of electrolytes at pH 6.0.Glutamate dehydrogenase, selectively desensitized by dinitrophenylation in the presence of ADP, can be activated by ADP at pH 9.0, but is no longer inhibited by the same effector at pH 6.0, high salt concentration. The densensitized enzyme is not inhibited by GTP at pH 9.0, but is activated by this effector at pH 6.0 in the presence of ionic compounds. Conversely, GTP-protected dinitrophenylated glutamate dehydrogenase is desensitized only to the effect of the activating modifier, ADP at pH 9.0, GTP at pH 6.0, high salt concentration. These findings suggest that the conformation of each allosteric site of glutamate dehydrogenase is changed by pH and ionic strength so that it keeps its specificity for the ligand which brings about a given effect, activation or inhibition, independently from its chemical structure.     

Cloning and characterization of gdhA, the structural gene for glutamate dehydrogenase of Salmonella typhimurium.

Glutamic acid is synthesized in enteric bacteria by either glutamate dehydrogenase or by the coupled activities of glutamate synthase and glutamine synthetase. A hybrid plasmid containing a fragment of the Salmonella typhimurium chromosome cloned into pBR328 restores growth of glutamate auxotrophs of S. typhimurium and Escherichia coli strains which have mutations in the genes for glutamate dehydrogenase and glutamate synthase. A 2.2-kilobase pair region was shown by complementation analysis, enzyme activity measurements, and the maxicell protein synthesizing system to carry the entire glutamate dehydrogenase structural gene, gdhA. Glutamate dehydrogenase encoded by gdhA carried on recombinant plasmids was elevated 5- to over 100-fold in S. typhimurium or E. coli cells and was regulated in both organisms. The gdhA promoter was located by recombination studies and by the in vitro fusion to, and activation of, a promoter-deficient galK gene. Additionally, S. typhimurium gdhA DNA was shown to hybridize to single restriction fragments of chromosomes from other enteric bacteria and from Saccharomyces cerevisiae.     

Affinity labeling of an allosteric ADP site of glutamate dehydrogenase by 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-monophosphate

Bovine liver glutamate dehydrogenase reacts covalently with the adenine nucleotide analogue 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-monophosphate (2-BDB-TAMP) with incorporation of about 1 mol of reagent/mol of enzyme subunit. The modified enzyme is not inactivated by this reaction as measured in the absence of allosteric effectors. Native glutamate dehydrogenase is activated by ADP and inhibited by high concentrations of NADH; both of these effects are irreversibly decreased upon reaction of the enzyme with 2-BDB-TAMP. The decrease in activation by ADP was used to determine the rate constant for reaction with 2-BDB-TAMP. The rate constant (kobs) for loss of ADP activation exhibits a nonlinear dependence on 2-BDB-TAMP concentration, suggesting a reversible binding of reagent (KR = 0.74 mM) prior to irreversible modification. At 1.2 mM 2-BDB-TAMP, kobs = 0.060 min-1 and is not affected by alpha-ketoglutarate or GTP, but is decreased to 0.020 min-1 by 5 mM NADH and to zero by 5 mM ADP. Incorporation after incubation with 1.2 mM 2-BDB-TAMP for 1 h at pH 7.1 is 1.02 mol/mol enzyme subunit in the absence but only 0.09 mol/subunit in the presence of ADP. The enzyme protected with 5 mM ADP behaves like native enzyme in its activation by ADP and in its inhibition by NADH. Native enzyme binds reversibly 2 mol of [14C]ADP/subunit, whereas modified enzyme binds only 1 mol of ADP/peptide chain. These results indicate that incorporation of 1 mol of 2-BDB-TAMP causes elimination of one of the ADP sites of the native enzyme. 2-BDB-TAMP acts as an affinity label of an ADP site of glutamate dehydrogenase and indirectly influences the NADH inhibitory site.     

5'-p-fluorosulfonyl)benzoyl-8-azidoadenosine: a new bifunctional affinity label for nucleotide binding sites in proteins

A new bifunctional affinity label, 5'-p-(fluorosulfonyl)benzoyl-8-azidoadenosine (5'-FSBAzA), has been synthesized by condensation of p-(fluorosulfonyl)benzoyl chloride with 8-azidoadenosine. 5'-FSBAzA has been characterized by elemental analysis, thin-layer chromatography, and ultraviolet and 1H NMR spectroscopy. The affinity label contains both an electrophilic fluorosulfonyl moiety and a photoactivatable azido group which are capable of reacting with several classes of amino acids found in enzymes. 5'-FSBAzA reacts with bovine liver glutamate dehydrogenase in a two-step process: a dark reaction yielding about 0.5 mol of the sulfonylbenzoyl-8-azidoadenosine (SBAzA) group bound/mol enzyme subunit by reaction of the enzyme at the fluorosulfonyl group, followed by photolysis in which 25% of the covalently bound SBAzA becomes crosslinked to the enzyme. 5'-FSBAzA-modified glutamate dehydrogenase, both before and after photolysis, retains full catalytic activity but is less sensitive to allosteric inhibition by GTP, to activation by ADP, and to inhibition by 1 mM NADH. These results suggest the modification in the dark reaction of a regulatory nucleotide binding site. Photoactivation of the covalently bound reagent may have general applicability in relating modified amino acids which are close to each other in the region of the purine nucleotide binding sites of glutamate dehydrogenase and other proteins.     

Binding studies of a spin-labelled oxidized coenzyme to bovine-liver glutamate dehydrogenase.

NAD+ with a nitroxide piperidine ring linked to the NH2 group of the adenine possesses full coenzymatic activity with glutamate dehydrogenase. Electron spin resonance spectra in the presence of glutamate dehydrogenase show mixtures of free and strongly immobilized spin-label. Binding studies in phosphate buffer demonstrate: (a) weak binary binding to the enzyme with a dissociation constant in the order of 2mM;(b) an indication for negative cooperativity or different sites for binding to enzyme-2-oxoglutarate, with dissociation constants in the order of 20--250muM; (c) similar but much weaker binding to enzyme-2-oxoglutarate-ADP; (d) a strong positive cooperative binding to enzyme-2-oxoglutarate-GTP, dependent on the enzyme concentration. Binding of phosphate to the enzyme with a Kd of about 20 mM or binding of pyrophosphate or tripolyphosphate with a Dd of about 2.5 mM enhances the binding of spin-labelled NAD+ in the presence of 2-oxoglutarate. There is evidence that the binding sites for these phosphates coincide with phosphate binding subsites of GTP.     

Immobilization of lactate dehydrogenase on polyacrylamide beads

Pig muscle lactate dehydrogenase (L-lactate:NAD oxidoreductase, EC 1.1.1.27) was covalently immobilized on polyacrylamide beads containing carboxylic functional groups activated by water-soluble carbodiimide. The effects of immobilization on the catalytic properties and stability of the lactate dehydrogenase were studied. There was no shift in the pH optimum of the immobilized enzyme compared to that of the soluble one. The apparent optimum temperature of the soluble enzyme was 65 degrees C, while that of the immobilized enzyme was between 50 and 65 degrees C. The apparent Km values of the immobilized enzyme with pyruvate and NADH substrates were higher than those of the soluble enzyme. As a result of immobilization, enhanced stabilities were found against heat treatment, changes in pH, and urea denaturation.     

Control of glutamate dehydrogenase from Pisum sativum roots

-Glutamate dehydrogenase (GDH) was found in soluble and particulate (mitochondrial) fractions of pea roots. The activity of NADH-dependent GDH in fresh mitochondrial extract was increased about 10-fold by addition of zinc, manganese or calcium, but high concentrations of zinc were inhibitory. During storage, GDH activity of the mitochondrial extract slowly increased. The NADH activity was inhibited by citrate and other chelating agents. NADH-dependent reductive amination was also inhibited by glutamate, the product of the reaction; by contrast NADPH dependent activity was relatively unaffected by zinc, chelating agents or glutamate. Sensitivity (of NADH-GDH) to glutamate was lost on purification, but was restored when the enzyme was immobilized by binding to an insoluble support (AE cellulose). Glutamate appears to change the affinity of the enzyme for 2-oxoglutarate.     

Spin-labeling studies of beef-liver glutamate dehydrogenase.

Bovine liver glutamate dehydrogenase was spin labeled with a nitroxide derivative of parachloromercuribenzoate. The ESR spectrum was of the immobilized type and the labeling yield 0.6 mole of spin label bound per mole of protomer under standard conditions. The specific activity of the labeled enzyme was not modified but the activation by ADP abolished. Inhibition by GTP was not altered but the ESR spectrum showed that the bound spin label was further immobilized in the presence of GTP and NADPH. In the presence of the coenzyme NADPH, the labeling yield decreased to half its initial value. Such a protection effect was observed neither with NADH nor with ADP.     

Effects of the changes in enzyme activities on metabolic flux redistribution around the 2-oxoglutarate branch in glutamate production by<Emphasis Type="Italic"> Corynebacterium glutamicum</Emphasis>

An experimental method for metabolic control analysis (MCA) was applied to the investigation of a metabolic network of glutamate production by Corynebacterium glutamicum. A metabolic reaction (MR) model was constructed and used for flux distribution analysis (MFA). The flux distribution at a key branch point, 2-oxoglutarate, was investigated in detail. Activities of isocitrate dehydrogenase (ICDH), glutamate dehydrogenase (GDH), and 2-oxoglutarate dehydrogenase complex (ODHC) around this the branch point were changed, using two genetically engineered strains (one with enhanced ICDH activity and the other with enhanced GDH activity) and by controlling environmental conditions (i.e. biotin-deficient conditions). The mole flux distribution was determined by an MR model, and the effects of the changes in the enzyme activities on the mole flux distribution were compared. Even though both GDH and ICDH activities were enhanced, the mole flux distribution was not significantly changed. When the ODHC activity was attenuated, the flux through ODHC decreased, and glutamate production was markedly increased. The flux control coefficients of the above three enzymes for glutamate production were determined based on changes in enzyme activities and the mole flux distributions. It was found that the factor with greatest impact on glutamate production in the metabolic network was obtained by attenuation of ODHC activity.     

Immobilization of horse liver alcohol dehydrogenase on copolymers of glycidyl methacrylate and ethylene dimethacrylate

Alcohol dehydrogenase has been immobilized to the basic copolymer and its several derivatives using various techniques. Enzyme coupling to the supports with amino groups by means of glutaraldehyde was found the most suitable. Activity of alcohol dehydrogenase coupled to these amino supports was comparable to that of the enzyme bound to Sepharose. Thermal and pH stability of alcohol dehydrogenase increased essentially upon immobilization. Kinetic properties of the immobilized enzyme differed from those of free alcohol dehydrogenase, pH optimum shifted to alkaline range, and apparent Michaelis constants for substrates and coenzymes increased. Curvatures observed in Lineweaver-Burk plots for coenzymes suggest an involvement of diffusion effects in the reaction catalyzed by alcohol dehydrogenase linked to these polymers.     

Immobilized phospholipase A2 from cobra venom. Prevention of substrate interfacial and activator effects

The activation of cobra venom phospholipase A2 by activators (containing phosphorylcholine moieties) appears to depend upon the aggregation state of the enzyme, and the presence of a lipid-water interface. The characteristics of this activation were studied by comparing the behavior of the enzyme immobilized on an agarose gel to that of the soluble enzyme. The immobilized enzyme displays only a few per cent of the soluble enzyme activity toward micellar dipalmitoyl-phosphatidylcholine (PC). However, the relative loss of activity is much less with micellar dipalmitoylphosphatidylethanolamine or soluble diheptanoyl-PC. The affinity for Ca2+ is increased about 10-fold by immobilization while the apparent pKa of the enzyme is decreased by 0.5-0.8 pH units. Activation energies are similar for the two enzyme forms and are independent of the physical state of the substrate used. Catalytic constants of the enzyme toward monomeric PC are not changed by immobilization. Yet, activators of the soluble enzyme have negligible effect on the immobilized enzyme, either in the presence or absence of an interface. Monomeric activators promote the binding of the soluble enzyme to the immobilized form. Apparently, immobilization mainly produces monomerically constrained enzyme which cannot be activated under any condition, whereas normally, activators in the presence of lipid-water interfaces induce the formation of enzyme dimers or possibly higher order aggregates.     

Immobilization of glyceraldehyde-3-phosphate dehydrogenase by non-covalent binding to specific antibodies and Fab-fragments coupled to Sepharose]

Rabbit antibodies to rat skeletal muscle glyceraldehyde-3-phosphate dehydrogenase, as well as monovalent Fab fragments of these antibodies were coupled to CNBr-activated Sepharose 4B. Rat skeletal muscle glyceraldehyde-3-phosphate dehydrogenase was then immobilized on a matrix by non-covalent binding to specific antibodies. Immobilized enzyme retains approximately 90% catalytic activity of the soluble dehydrogenase; pH optimum of activity and the Km value observed are changed as compared to the enzyme in solution. Glyceraldehyde-3-phosphate dehydrogenase immobilized on specific antibodies is shown to undergo adenine nucleotide-induced dissociation into dimers. The immobilized dimeric form of the enzyme thus obtained is catalytically active and capable of reassociating with the dimers of apoglyceraldehyde-3-phosphate dehydrogenase added in solution to the suspension of Sepharose.