The invasive adenylate cyclase of Bordetella pertussis. Intracellular localization and kinetics of penetration into various cells.

The penetration of Bordetella pertussis adenylate cyclase into various mammalian cells exhibits similar kinetics; the accumulation of both intracellular cyclase activity and cyclic AMP is rapid, reaching constant levels after 15-60 min of incubation. The kinetics of enzyme penetration into turkey erythrocytes is different; cyclase activity and cyclic AMP accumulate linearly and do not reach constant levels even after 6 h of incubation. In the preceding paper [Friedman, Farfel & Hanski (1987) Biochem. J. 243, 145-151] we have suggested that the constant level of intracellular cyclase activity reflects a steady state formed by continuous penetration and intracellular inactivation of the enzyme. In contrast with other mammalian cells, no inactivation of cyclase is observed in turkey erythrocytes. These results further support the notion that there is continuous penetration and deactivation of the invasive enzyme in mammalian cells. A 5-6-fold increase in specific activity of the invasive cyclase is detected in a pellet fraction of human lymphocytes in which a similar increase in specific activity of the plasma-membrane marker 5'-nucleotidase is observed. A similar increase in the invasive-cyclase specific activity is detected in a membrane fraction of human erythrocytes. Cyclase activity in a membrane-enriched fraction of human lymphocytes reached a constant level after 20 min of cell exposure to the enzyme. Similar time courses were observed for accumulation of cyclase activity and cyclic AMP in whole lymphocytes [Friedman, Farfel & Hanski (1987) Biochem, J. 243, 145-151]. We suggest therefore that cyclic AMP generation by the invasive enzyme as well as the intracellular inactivation process occur while it is associated with a membrane fraction identical, or closely associated, with the plasma membrane.     

Interaction of ferredoxin and ferredoxin-NADP reductase with thylakoids

Ferredoxin-NADP reductase accounts for about 50% of the NADPH diaphorase activity of spinach leaf homogenates. The enzyme is bound to thylakoid membranes, but can be slowly extracted by aqueous buffers. Ferredoxin-NADP reductase can be extracted from the membranes by a 1- to 2-min treatment with a low concentration of trypsin. This treatment completely inactivates NADP photoreduction but does not affect electron transport from water to ferredoxin. It is shown that the inactivation is due to solubilization of ferredoxin-NADP reductase: the activity can be restored by addition of a very large excess of soluble enzyme in pure form. When ferredoxin-NADP reductase is added as a soluble enzyme after extraction or inactivation (by a specific antibody) of the membrane-bound enzyme, NADP photoreduction requires a very large excess of this enzyme, and the apparent K_m for ferredoxin is also increased. These observations are discussed as related to the interactions of thylakoids with ferredoxin-NADP reductase.     

Inactivation of rat ovarian 20α-hydroxysteroid dehydrogenase by N-alkylmaleimides

A series of N-alkylmaleimides, varying in chain length from N-ethylmaleimide and N-butyl to N-octyl, inclusive, was shown to effectively inactivate rat ovarian 20α-hydroxysteroid dehydrogenase at pH 7.7, 25 °C. The apparent second-order rate constants for inactivation were observed to increase with increasing chain length of the N-alkylmaleimide used. Positive chain length effects were also indicated by the K_d values for N-alkylmaleimides calculated from double-reciprocal plots resulting from the saturation kinetics observed in the inactivation reactions. The maximum rate constant for inactivation at enzyme saturation was 0.3 min^?1 for each maleimide studied. NADP-and coenzyme-competitive inhibitors such as 3-aminopyridine adenine dinucleotide phosphate and various adenosine derivatives protected the enzyme against maleimide inactivation, whereas no protection was observed with the steroid substrate, 20α-hydroxypregn-4-en-3-one. The pH profile for maleimide inactivation indicated the involvement of an enzyme functional group with a pK_a near 8.0. Sulfhydryl modification was also indicated by fluorescein mercuric acetate inactivation and titration experiments. Inactivation of the enzyme by a lysine-modifying reagent exhibited a pH profile differing from that observed in the maleimide inactivation process. It is proposed that N-alkylmaleimides inactivate the enzyme through covalent modification of sulfhydryl groups located in a nonpolar region of the enzyme.     

Essential arginine residues in human liver arylsulfatase A.

Human liver arylsulfatase A was treated with arginine-specific reagents (diones), resulting in a loss of enzyme activitity with apparent first-order kinetics. Sulfite and borate—competitive inhibitors of the enzyme—provided complete protection from inactivation by phenylglyoxal. Sulfite and substrate each likewise protected against enzyme inactivation by 2,3-butanedione. A plot of pseudo-first-order rate constants of enzyme inactivation versus 2,3-butanedione concentrations suggests that an essential arginine residue is modified with a loss in function of the binding site or of the active site of the protein. Chemical analysis of the butanedione-treated sulfatase indicates that complete enzyme inactivation corresponds to a modification of only about 2 of the 20 arginine residues per enzyme subunit. Taken together, all of the results strongly suggest that arginine residues are essential for the activity of arylsulfatase A. An incidental discovery in this work is that borate ion is a competitive inhibitor of human arylsulfatase A with a K_i of 2.5 × 10^?4 M.     

Inactivation of nitric oxide by cytochrome c oxidase under steady-state oxygen conditions

We have developed a respiration chamber that allows intact cells to be studied under controlled oxygen (O₂) conditions. The system measures the concentrations of O₂ and nitric oxide (NO) in the cell suspension, while the redox state of cytochrome c oxidase is continuously monitored optically. Using human embryonic kidney cells transfected with a tetracycline-inducible NO synthase we show that the inactivation of NO by cytochrome c oxidase is dependent on both O₂ concentration and electron turnover of the enzyme. At a high O₂ concentration (70 μM), and while the enzyme is in turnover, NO generated by the NO synthase upon addition of a given concentration of l-arginine is partially inactivated by cytochrome c oxidase and does not affect the redox state of the enzyme or consumption of O₂. At low O₂ (15 μM), when the cytochrome c oxidase is more reduced, inactivation of NO is decreased. In addition, the NO that is not inactivated inhibits the cytochrome c oxidase, further reducing the enzyme and lowering O₂ consumption. At both high and low O₂ concentrations the inactivation of NO is decreased when sodium azide is used to inhibit cytochrome c oxidase and decrease electron turnover.     

Inactivation of chicken liver d-3-phosphoglycerate dehydrogenase by N-alkylmaleimides

Chicken liver d-3-phosphoglycerate dehydrogenase was effectively inhibited at 25 °C by micromolar concentrations of N-ethyl-, N-butyl-, N-pentyl-, N-heptyl-, and N-phenylmaleimide. The rates of inactivation of the enzyme did not vary with chain length of the N-alkylmaleimide derivative. Saturation kinetics in the same concentration range was observed with each maleimide derivative studied. A maximum pseudo-first-order rate constant of 0.1 min^?1 was determined for all of the maleimide inactivation reactions. Compounds shown to bind at the coenzyme binding site such as NAD, 3-aminopyridine adenine dinucleotide, adenosine diphosphoribose, and adenosine diphosphate did not protect the enzyme against N-ethylmaleimide inactivation. AMP was demonstrated to be a substrate-competitive inhibitor of the enzyme. AMP and 3-phosphoglycerate both effectively protected the enzyme against N-ethylmaleimide inactivation. Diazotized 3-aminopyridine adenine dinucleotide, a sulfhydryl modifying, site-labeling reagent for several pyridine nucleotide-dependent enzymes, did not inactivate the phosphoglycerate dehydrogenase but functioned rather as a reversible coenzyme-competitive inhibitor.     

Modification of three enzymes of the β-ketoadipate pathway by N-methyl-N′-nitro-N-mtrosoguanidine

The sensitivities of three enzymes of the β-ketoadipate pathway to inactivation by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) were determined in vivo and in vitro under conditions compatible with mutagenesis.One enzyme, β-ketoadipate enol-lactone hydrolase, is very sensitive to inactivation by low concentrations of MNNG. This enzyme is also sensitive to inactivation by N-ethylmaleimide and mercurial reagents. The free sulfhydryl content of native enol-lactone hydrolase was determined to be two moles free sulfhydryl per mole of enzyme. A 95% inactivation of enol-lactone hydrolase by MNNG results in a masking of slightly more than one mole sulfhydryl per mole enzyme.Muconate lactonizing enzyme is moderately sensitive to inactivation by low concentrations of MNNG, but is not inactivated by sulfhydryl reagents. Muconolactone isomerase is resistant to inactivation by low concentrations of MNNG and is not inactivated by sulfhydryl reagents. Upon exposure to high concentrations of MNNG, muconolactone isomerase is rapidly inactivated. Spectrophotometric evidence indicates the lysine residues are nitroguanidinated proportionally with a loss in the enzymatic activity.These data indicate that the exposure of cells to low concentrations of MNNG should affect the activity of enzymes with essential sulfhydryl groups.     

Genetic Modification of the Penicillin G Acylase Surface To Improve Its Reversible Immobilization on Ionic Exchangers

A new mutant of the industrial enzyme penicillin G acylase (PGA) from Escherichia coli has been designed to improve its reversible immobilization on anionic exchangers (DEAE- or polyethyleneimine [PEI]-coated agarose) by assembling eight new glutamic residues distributed homogeneously through the enzyme surface via site-directed mutagenesis. The mutant PGA is produced and processed in vivo as is the native enzyme. Moreover, it has a similar specific activity to and shows the same pH activity profile as native PGA; however, its isoelectric point decreased from 6.4 to 4.3. Although the new enzyme is adsorbed on both supports, the adsorption was even stronger when supports were coated with PEI, allowing us to improve the enzyme stability in organic cosolvents. The use of restrictive conditions during the enzyme adsorption on anionic exchangers (pH 5 and high ionic strength) permitted us to still further increase the strength of adsorption and the enzyme stability in the presence of organic solvents, suggesting that these conditions allow the penetration of the enzyme inside the polymeric beds, thus becoming fully covered with the polymer. After the enzyme inactivation, it can be desorbed to reuse the support. The possibility to improve the immobilization properties on an enzyme by site-directed mutagenesis of its surface opens a promising new scenario for enzyme engineering.     

Irreversible inactivation of rat liver tyrosine aminotransferase

Homogenates prepared from rat livers irreversibly inactivate tyrosine aminotransferase, both endogenous and purified exogenous enzyme, in the presence of certain compounds which bind to pyridoxal 5′-P. The rate of inactivation ranged from a half-life of 0.72 to greater than 15 hr. The pyridoxal 5′-P binding compounds may be considered to be structural analogs for α-ketoglutarate or l-tyrosine, both of which are substrates for the enzyme. l-Cysteine and l-DOPA are the most effective compounds tested of each of the two structural analog classes, respectively. Absence of the carboxyl group from l-cysteine or l-DOPA has little effect on the half-life of the enzyme, whereas absence or substitution of the amino group results in an increased enzyme half-life. Absence of the —SH group from l-cysteine or of the 3′-OH group from l-DOPA results in little or no inactivation of the enzyme ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ increased to greater than 15 hr). Semicarbazide and hydroxylamine have little effect on the stability of the enzyme. Addition of pyridoxal 5′-P to homogenates incubated with l-cysteine or l-DOPA inhibits the inactivation of the enzyme. However, the addition of cofactor to inactivated enzyme does not restore lost activity.There is a disappearance of antigenic cross-reacting material during inactivation of the enzyme. This loss of specific cross-reacting material occurs at a slower rate than the loss of enzyme activity, indicating that enzymatic activity is lost prior to loss of antigenic recognition. A three-step proposal is presented to explain the data observed in which the first step is a reversible loss of pyridoxal 5′-P from the enzyme, followed by a specific irreversible inactivation of the enzyme, and ending with nonspecific proteolysis or degradation of the inactivated enzyme molecules.     

The reaction of alkylating agents with cyclic 3',5'-nucleotide phosphodiesterase

The reaction of anti-tumour alkylating agents with beef heart cyclic nucleotide phosphodiesterase (adenosine 3',5'-monophosphate phosphohydrolase, EC 3.1.4.c) has been investigated. This enzyme exists in two forms differing in their Michaelis-Menten K_m values. Chlorambucil [p-(di-2-chloroethylamino)-phenyl-butyric acid] inhibits the form of the enzyme with a low K_m value with a velocity constant for inactivation three times that for inhibition of the high K_m form. While the monofunctional N-ethyl analogue of chlorambucil is ineffective as an inhibitor of either form of the enzyme, iodoacetate inhibits both forms, though the velocity constant for inactivation of each form is much less than that for chlorambucil. Also the rate of inactivation of each form does not significantly differ. A cross-linking mechanism for the inactivation of regulatory enzymes is proposed.     

Purification and characterization of a zearalenone degrading enzyme produced by<Emphasis Type="Italic">Gliocladium roseum</Emphasis>

Enzymatic inactivation of fungal toxins is an attractive strategy for the decontamination of food and feeding stuff. A constitutively expressed enzyme opening the lactone linkage within the macrocyclic ring system of zearalenone (ZON) was isolated fromGliocladium roseum. The enzyme has been shown to catalyze the transformation of the mycotoxin ZON and therefore has been named ZON degrading enzyme. The resulting products of the enzymatic reaction are less toxic because they have lost their estrogenic capacity. In this study, we used scanning electron microscopy to evaluate the possible mycoparasitism betweenFusarium graminearum andG. roseum. The ZON-degrading enzyme could be isolated fromG. roseum cultures and biochemically characterized. It has been found to be similar to superoxide-dismutases at its N-teminus.     

Cold-active esterase from Psychrobacter sp. Ant300: gene cloning,characterization, and the effects of Gly→Pro substitution near the active site on its catalytic activity and stability

The gene encoding an esterase (PsyEst) of Psychrobacter sp. Ant300, a psychrophilic bacterium isolated from Antarctic soil, was cloned, sequenced, and expressed in Escherichia coli. PsyEst, which is a member of hormone-sensitive lipase (HSL) group of the lipase/esterase family, is a cold-active, themolabile enzyme with high catalytic activity at low temperatures (5–25 °C), low activation energy (e.g., 4.6 kcal/mol for hydrolysis of p-nitrophenyl butyrate), and a t_1/2 value of 16 min for thermal inactivation during incubation at 40 °C and pH 7.9. A three-dimensional structural model of PsyEst predicted that Gly²⁴⁴ was located in the loop near the active site of PsyEst and that substitution of this amino-acid residue by proline should potentially rigidify the active-site environment of the enzyme. Thus, we introduced the Gly²⁴⁴→Pro substitution into the enzyme. Stability studies showed that the t_1/2 value for thermal inactivation of the mutant during incubation at 40 °C and pH 7.9 was 11.6 h, which was significantly greater than that of the wild-type enzyme. The k_cat/K_m value of the mutant was lower for all substrates examined than the value of the wild type. Moreover, this amino-acid substitution caused a shift of the acyl-chain length specificity of the enzyme toward higher preference for short-chain fatty acid esters. All of these observations could be explained in terms of a decrease in active-site flexibility brought about by the mutation and were consistent with the hypothesis that cold activity and thermolability arise from local flexibility around the active site of the enzyme.     

Kinetics of Inhibition of Green Crab (Scylla serrata) Alkaline Phosphatase by L-Cysteine

The inhibition of alkaline phosphatase from green crab (Scylla serrata) by L-cysteine has been studied. The results show that L-cysteine gives a mixed-type inhibition. The progress-of-substrate-reaction method previously described by Tsou [(1988), Adv. Enzymol. Related Areas Mol. Biol. 61, 391–436] was used to study the inactivation kinetics of the enzyme by L-cysteine. The microscopic rate constants were determined for reaction of the inhibitor with the free enzyme and the enzyme–substrate complex (ES) The results show that inactivation of the enzyme by L-cysteine is a slow, reversible reaction. Comparison of the inactivation rate constants of free enzyme and ES suggests that the presence of the substrate offers marked protection of this enzyme against inactivation by L-cysteine.     

Modification of the active site of isocitrate lyase from Pseudomonas indigofera

Kinetic analysis of inactivation of isocitrate lyase from Pseudomonas indigofera by 3-bromopyruvate established that enzyme binds this compound prior to alkylation and that substrate, D_s-isocitrate, competes for the same site on the enzyme. The rate of inactivation was increased by EDTA which is a promoter of catalysis in the presence of activated (reduced) enzyme and substrate. The combination of products, glyoxylate plus succinate, also protected against inactivation. Glyoxylate plus itaconate, phosphoenolpyruvate, or maleate also protected. However, each of the latter three compounds or glyoxylate or succinate alone provided little or no protection. Pyruvate, a competitive inhibitor with respect to glyoxylate in the condensation reaction, also failed to protect. However, two dicarboxylates, meso-tartrate and oxalate, that are also competitive inhibitors with respect to glyoxylate provide some protection against inactivation by BrP perhaps by bridging across cationic sites that facilitate glyoxylate and succinate binding. These and other results imply that alkylation by 3-bromopyruvate occurs at the succinate part of the active site. A mechanism which includes a catalytic role for the cysteine residue at the active site is presented and discussed.     

Cold-lability of prolyl-tRNA synthetase from higher plants

Pro-tRNA synthetase from D. regia and P. aureus lost enzymic activity more rapidly at 0° than at room temperature. The enzyme from a number of higher plants that produce azetidine-2-carboxylic acid (A2C) was more rapidly inactivated in the cold than the enzyme from plants which do not contain A2C. The rate of cold inactivation was dependent on temperature and on the concentration of glycerol, protein and sulphydryl-reducing reagents. Substrates of Pro-tRNA synthetase also stabilized the enzyme against cold inactivation. Enzyme which had been completely inactivated by storage in the cold, could be reactivated by warming in the presence of a sulphydryl-reducing reagent. The rate of reactivation was dependent on temperature, pH and the concentration of sulphydryl-reducing reagent. Kinetic analysis indicated the existence of more than one molecular form of the enzyme. It is suggested that the cold-lability of Pro-tRNA synthetase may be due to dissociation of the active enzyme molecule into inactive subunits.     

Evidence for the Existence of Two Essential and Proximal Cysteinyl Residues in NADP-Malic Enzyme from Maize Leaves

Incubation of maize (Zea mays) leaf NADP-malic enzyme with monofunctional and bifunctional N-substituted maleimides results in an irreversible inactivation of the enzyme. Inactivation by the monofunctional reagents, N-ethylmaleimide (NEM) and N-phenylmaleimide, followed pseudo-first-order kinetics. The maximum inactivation rate constant for phenylmaleimide was 10-fold higher than that for NEM, suggesting a possible hydrophobic microenvironment of the residue(s) involved in the modification of the enzyme. In contrast, the inactivation kinetics with the bifunctional maleimides, ortho-, meta-, and para-phenylenebismaleimide, were biphasic, probably due to different reactivities of the groups reacting with the two heads of these bifunctional reagents, with a possible cross-linking of two sulfhydryl groups. The inactivation by mono and bifunctional maleimides was partially prevented by Mg²⁺ and l-malate, and NADP prevented the inactivation almost totally. Determination of the number of reactive sulfhydryl groups of the native enzyme with [³H]NEM in the absence or presence of NADP showed that inactivation occurred concomitantly with the modification of two cysteinyl residues per enzyme monomer. The presence of these two essential residues was confirmed by titration of sulfhydryl groups with [³H]NEM in the enzyme previously modified by o-phenylenebismaleimide in the absence or presence of NADP.     

Inactivation of maize leaf phosphoenolpyruvate carboxylase by the binding to chloroplast membranes

Phosphoenolpyruvate carboxylase (PEPC) purified from maize (Zea mays L.) leaves associates with maize leaf chloroplast membrane in vitro. The binding of PEPC to the membrane results in enzyme inactivation. A protein isolated from a maize leaf chloroplast membrane preparation inactivated PEPC. Treatment with membrane preparation or with partially purified inactivating protein accelerates PEPC inactivation at low temperature (4°C). Interaction of PEPC with chloroplast membrane or inactivating protein may inactivate the enzyme by influencing dissociation of the enzyme active tetramer.     

Reversible inactivation of the O2-labile hydrogenases from Azotobacter vinelandii and Rhizobium japonicum

Hydrogenases catalyze the reversible activation of dihydrogen. The hydrogenases from the aerobic, N2-fixing microorganisms Azotobacter vinelandii and Rhizobium japonicum are nickel- and iron-containing dimers that belong to the group of O2-labile enzymes. Exposure of these hydrogenases to O2 results in an irreversible inactivation; therefore, these enzymes are purified anaerobically in a fully active state. We describe in this paper an electron acceptor-requiring and pH-dependent, reversible inactivation of these hydrogenases. These results are the first example of an anaerobic, reversible inactivation of the O2-labile hydrogenases. The reversible inactivation required the presence of an electron acceptor. The rate of inactivation was first-order, with similar rates observed for methylene blue, benzyl viologen, and phenazine-methosulfate. The rate of inactivation was also dependent on the pH. However, increasing the pH of the enzyme in the absence of an electron acceptor did not result in inactivation. Thus, the reversible inactivation was not a result of high pH alone. The inactive enzyme could not be reactivated by H2 or other reductants at high pH. Titration of enzyme inactivated at high pH back to low pH was also ineffective at reactivating the enzyme. However, if reductants were present during this titration, the enzyme could be fully reactivated. The temperature dependence of inactivation yielded an activation energy of 44 kJ X mol-1. Gel filtration chromatography of active and inactive hydrogenase indicated that neither dissociation nor aggregation of the dimer hydrogenase was responsible for this reversible inactivation. We propose a four-state model to describe this reversible inactivation.     

The effect of γ-radiation on the NADP-MALIC enzyme from mango fruit,mangifera indica—I. Physico-chemical aspects

• 1.1. Malic enzyme purified from the fruit tissue of Mangifera indica was irradiated in dilute solution and the effect of γ-irradiation was investigated.
• 2.2. The activity of the enzyme decreased exponentially as a function of the applied dose under all conditions investigated. The inactivation yield (Go-value) in neutral solution and in air was 0.069.
• 3.3. The role of the radicals produced by water radiolysis in the inactivation of the enzyme was investigated by using different gas atmospheres and selective free radical-anions. The hydrogen atom and the hydrated electron (reducing species) were found to be important in the enzyme inactivation; as well as the possible destruction of cysteine and tryptophan residues.
• 4.4. The irradiated enzyme appears to adopt a more compact conformation as reflected in a slightly lower M_r, Stokes-radius and diffusion coefficient.
• 5.5. γ-Radiation does not lead to any heterogeneity in the charge and size properties of the enzyme and the pI and the M_r of the subunits were unaffected.
• 6.6. Some differences in the amino acid composition of the non-irradiated and irradiated enzyme were observed but specific amino acid residues were not preferentially destroyed.
• 7.7. These changes were also reflected in the ultraviolet spectrum of the enzyme which shifted to lower values.
• 8.8. The major cause of inactivation seem to be a change in conformation caused by chemical modification of amino acid side chains.

     

Thermal inactivation of a Citrobacter ribonuclease: Influence of polyamines and ionic strength

The thermal inactivation of a Citrobacter sp. ribonuclease (RNase) is subject to control by a number of factors. Low concentrations of naturally occurring polyamines such as spermidine and spermine, and certain analogs of these compounds, protect the enzyme from inactivation. Changes in ionic strength cause wide variations in the rate at which enzyme activity is lost. Additionally, depending on the type of ion added to the reaction mixture, the rate constant for enzyme inactivation-may either increase or decrease as the ionic strength is raised. Thermodynamic parameters were determined under a variety of experimental conditions for the thermal inactivation of this RNase. It was found in all of these cases that the entropy of activation is large and negative, implying that a gross change in enzyme conformation is not taking place. The concentration and identity of ions present and the amount of polyamine available to interact with this RNase determines the rate of loss, by thermal inactivation, of enzyme activity in this in vitro system. These factors therefore constitute a system whereby substrate hydrolysis may be controlled with time.