Reversible inactivation of tyrosine aminotransferase from guinea pig liver by thiol and disulfide compounds.

Tyrosine aminotransferase from guinea pig liver is strongly inactivated by a variety of natural thiols and disulfides. L-cysteine was used as a model compound in the study of inactivation. Inactivation is due to the disulfide produced by spontaneous oxidation of thiol during incubation. Binding studies with [³⁵S]-cysteine revealed simultaneous incorporation of [³⁵S] into tyrosine aminotransferase and loss of enzyme activity. The reversibility demonstrates that the inactivation is the result of the formation of mixed disulfide between the disulfide and the sulfhydryl group of tyrosine aminotransferase. Some features of the enzyme active site are showed by the inactivation reaction.     

Participation of cysteine and cystine in inactivation of tyrosine aminotransferase in rat liver homogenates.

1. Inactivation of tyrosine aminotransferase was studied in rat liver homogenates. Under an O2 atmosphere with cysteine added, inactivation was rapid after a lag period of approx. 1h, whereas a N2 atmosphere extended the lag period to approx. 3h. 2. Replacement of cysteine with cystine resulted in rapid inactivation both aerobically and anaerobically. 3. Removal of the particulate fraction by centrifuging rat liver homogenates at 13,000g for 9min resulted in an aerobic lag period of 0.5h in the presence of cystine and approx. 3h in the presence of cysteine. 4. It is proposed that the stimulatory effect of cysteine on tyrosine aminotransferase inactivation occurs largely as a result of oxidation to cystine, which appears to be a more directly effective agent.     

Stabilization of rat liver tyrosine aminotransferase by tetracycline.

Rat liver tyrosine aminotransferase was purified 200-fold and an antiserum raised against it in rabbits. 2. Hepatic tyrosine aminotransferase activity was increased fourfold by tyrosine, twofold by tetracycline, 2.5-fold by cortisone 21-acetate and ninefold by a combination of tyrosine and cortisol administered intraperitoneally to rats. 3. Radioimmunoassay with 14C-labelled tyrosine aminotransferase, in conjunction with rabbit antiserum against the enzyme, revealed that cortisol stimulates the synthesis of the enzyme de novo, but that tetracycline has no such effect. 4. Incubation of rat liver homogenates with purified tyrosine aminotransferase in vitro leads to a rapid inactivation of the enzyme, which tetracycline partially inhibits. 5. The inactivation is brought about by intact lysosomes, and the addition of 10mM-cysteine increases the rate of enzyme inactivation, which is further markedly increased by 10mM-Mg2+ and 10mM-ATP. Here again tetracycline partially inhibits the decay rate, leading to the inference that the increase of tyrosine aminotransferase activity in vivo by tetracycline is brought about by the latter inhibiting the lysosomal catheptic action.     

Persulfide generated from L-cysteine inactivates tyrosine aminotransferase. Requirement for a protein with cysteine oxidase activity and gamma-cystathionase

Liver cytosols contain factors that produce an inhibitor of tyrosine aminotransferase and other enzymes when incubated with L-cysteine or L-cystine. Cystine-dependent inactivation was caused by cystathionase and required pyridoxal 5'-phosphate, but a second protein was needed to reconstitute cysteine-dependent inactivation. A cytosolic protein was isolated that oxidized free cysteine and brought about inactivation of tyrosine aminotransferase when coincubated with cystathionase. Hematin also oxidized cysteine, which led to cysteine-dependent inactivation of tyrosine aminotransferase in the presence of cystathionase. The inactivation of tyrosine aminotransferase involved three steps: initial oxidation of cysteine to form cystine; desulfuration of cystine catalyzed by cystathionase to form the persulfide, thiocysteine; and reaction of thiocysteine (or products of its decomposition) with proteins to form protein-bound sulfane. Since dithiothreitol reactivated tyrosine aminotransferase, the sulfane probably inactivated the enzyme by oxidation of thiol groups. The present results do not indicate whether the cysteine oxidase activity is enzymatic nor do they prove which form of polysulfide inactivates tyrosine aminotransferase. Reduced glutathione greatly slowed the rates at which sulfane accumulated and at which tyrosine aminotransferase was inactivated. Incubation of DL-cystathionine with liver cytosols led to formation of cysteine, which was oxidized and cleaved to form persulfide, and caused inactivation of tyrosine aminotransferase. Thus, sulfane sulfur that is generated by an enzyme of the transulfuration pathway inactivates a transaminase by nonselective oxidation of enzyme-bound thiol groups.     

Effect of physiological reducing compounds on the inactivation of tyrosine aminotransferase from guinea-pig liver.

1. Tyrosine aminotransferase from guinea-pig liver is inactivated at neutral pH by a factor localized in the microsomal fraction. The inactivation, independent of exogenous L-cysteine, is rapidly reversed by addition of dithiothreitol. 2. The effects of physiological reducing agents on the enzyme inactivation were investigated. L-Cysteine and L-cysteamine enhance the inactivation rate of the enzyme in the presence of microsomal membranes, and also they are able to bring about the loss in enzyme activity independently of microsomal action. Reduced glutathione, at physiological concentration, and NADPH decrease the inactivation rate. Other physiological reducing compounds, as well as oxidized glutathione and NADP+, are without effect. 3. Neither reduced glutathione nor NADPH, unlike dithiothreitol and mercaptoethanol, is able to restore the activity of partially inactivated tyrosine aminotransferase. 4. It is proposed that the intracellular concentration of reduced glutathione might modulate the rate of inactivation of the enzyme in vivo.     

Inactivation of gamma-aminobutyric acid aminotransferase by (Z)-4-amino-2-fluorobut-2-enoic acid

(Z)-4-Amino-2-fluorobut-2-enoic acid (1) is shown to be a mechanism-based inactivator of pig brain gamma-aminobutyric acid aminotransferase. Approximately 750 inactivator molecules are consumed prior to complete enzyme inactivation. Concurrent with enzyme inactivation is the release of 708 +/- 79 fluoride ions; transamination occurs 737 +/- 15 times per inactivation event. Inactivation of [3H]pyridoxal 5'-phosphate ([3H]PLP) reconstituted GABA aminotransferase by 1 followed by denaturation releases [3H]PMP with no radioactivity remaining attached to the protein. A similar experiment carried out with 4-amino-5-fluoropent-2-enoic acid [Silverman, R. B., Invergo, B. J., & Mathew, J. (1986) J. Med. Chem. 29, 1840-1846] as the inactivator produces no [3H]PMP; rather, another radioactive species is released. These results support an inactivation mechanism for 1 that involves normal catalytic isomerization followed by active site nucleophilic attack on the activated Michael acceptor. A general hypothesis for predicting the inactivation mechanism (Michael addition vs enamine addition) of GABA aminotransferase inactivators is proposed.     

Chemical and kinetic evidence for an essential histidine in horseradish peroxidase for iodide oxidation.

Horseradish peroxidase (HRP), when incubated with diethylpyrocarbonate (DEPC), shows a time-dependent loss of iodide oxidation activity. The inactivation follows pseudo-first order kinetics with a second order rate constant of 0.43 min-1 M-1 at 30 degrees C and is reversed by neutralized hydroxylamine. The difference absorption spectrum of the modified versus native enzyme shows a peak at 244 nm, characteristic of N-carbethoxyhistidine, which is diminished by treatment with hydroxylamine. Correlation between the stoichiometry of histidine modification and the extent of inactivation indicates that out of 2 histidine residues modified, one is responsible for inactivation. A plot of the log of the reciprocal half-time of inactivation against log DEPC concentration further suggests that only 1 histidine is involved in catalysis. The rate of inactivation shows a pH dependence with an inflection point at 6.2, indicating histidine derivatization by DEPC. Inactivation due to modification of tyrosine, lysine, or cysteine has been excluded. CD studies reveal no significant change in the protein or heme conformation following DEPC modification. We suggest that a unique histidine residue is required for maximal catalytic activity of HRP for iodide oxidation.     

Kinetics of inactivation of beta-lactamase I by 6 beta-bromopenicillanic acid.

The kinetics of the inactivation of beta-lactamase I from Bacillus cereus 569 by preparations of 6 alpha-bromopenicillanic acid showed unexpected features. These can be quantitatively accounted for on the basis of the inactivator being the epimer, 6 beta-bromopenicillanic acid. At pH 9.2, the rate-determining step in the inactivation is the formation of the inactivator. When pure 6 beta-bromopenicillanic acid is used to inactivate beta-lactamase I, simple second-order kinetics are observed. The inactivated enzyme has a new absorption peak at 326 nm. The rate constant for inactivation has the same value as the rate constant for appearance of absorption at 326 nm; the rate-determining step may thus be fission of the beta-lactam ring of 6 beta-bromopenicillanic acid. Inactivation is slower in the presence of substrate, and the observed kinetics can be quantitatively accounted for on a simple competitive model. The results strongly suggest that inactivation is a consequence of reaction at the active site.     

Acid inactivation of short-lived rat liver enzymes.

The stabilities of nine rat liver cytosol enzymes were compared at a variety of pH values. The cytosol enzymes studied were (a) those with half-lives in vivo of 3 days or longer: lactate dehydrogenase, arginase, glyceraldehyde phosphate dehydrogenase and alanine aminotransferase, (b) those with half-lives in vivo shorter than 2 days; glucokinase, dihydroorotase, serine dehydratase and tyrosine aminotransferase and (c) catalase, which has an intermediate half-life of 2.5 days for the protein protion. All the enzymes were stable in vitro at neurtal and alkaline pH values. However, at acidic pH values (pH 4): the long-lived enzymes (a) were stable; the short-lived enzymes (b) were completely inactivated with one exception; and catalase was partially inactivated. Tyrosine aminotransferase was the exception in that it is a short-lived enzyme in vivo but stable under all conditions tested in vitro. The finding that long-lived enzymes are stable in an acid milieu and short-lived enzymes are generally unstable was only observed if certain ligands (NAD+, pyridoxal 5'-phosphate, Mn2+, amino acids) were added to the invitro system. Lysosomal extracts did not accelerate the rate of inactivation of any cytosol enzyme in acidic solutions. These results indicate that if degradation of intracellular enzymes occurs in lysosomes, acid inactivation and denaturation of enzymes may be the initial event in determining the functional half-lives of the enzymes in vivo.     

Inactivation of pig heart alanine aminotransferase by beta-chloroalanine.

The substrate analog β-chloro-l-alanine rapidly inactivates the pig heart alanine aminotransferase. Inactivation is dependent upon formation of an intermediate. The substrate alanine protects the enzyme by competing with the inhibitor for the formation of this intermediate. Halide and carboxylate anions accelerated the rate of inactivation once the enzyme-inhibitor complex was formed. This acceleration appears to mimic the action with the substrate, for the rate of exchange transamination between unlabeled alanine and labeled pyruvate is similarly accelerated. When labeled inhibitor was used, the inactive enzyme became labeled. The spectral changes which occur resemble, in many respects, those which occur with aspartate aminotransferase when its active-site lysine undergoes alkylation by β-chloroalanine. We conclude that chloroalanine fulfills the criteria for a “suicide substrate.”     

Acid inactivation of short-lived rat liver enzymes

The stabilities of nine rat liver cytosol enzymes were compared at a variety of pH values. The cytosol enzymes studied were (a) those with half-lives in vivo of 3 days or longer: lactate dehydrogenase, arginase, glyceraldehyde phosphate dehydrogenase and alanine aminotransferase, (b) those with half-lives in vivo shorter than 2 days; glucokinase, dihydroorotase, serine dehydratase and tyrosine aminotransferase and (c) catalase, which has an intermediate half-life of 2.5 days for the protein portion. All the enzymes were stable in vitro at neutral and alkaline pH values. However, at acidic pH values (pH 4): the long-lived enzymes (a) were stable; the short-lived enzymes (b) were completely inactivated with one exception; and catalase was partially inactivated. Tyrosine aminotransferase was the exception in that it is a short-lived enzyme in vivo but stable under all conditions tested in vitro. The finding that long-lived enzymes are stable in an acid milieu and short-lived enzymes are generally unstable was only observed if certain ligands (NAD⁺, pyridoxal 5′-phosphate, Mn²⁺, amino acids) were added to the iv vitro systems. Lysosomal extracts did not accelerate the rate of inactivation of any cytosol enzyme in acidic solutions. These results indicate that if degradation of intracellular enzymes occurs in lysosomes, acid inactivation and denaturation of enzymes may be the initial event in determining the functional half-lives of the enzymes in vivo.     

Effect of concanavalin A on tyrosine aminotransferase in rat hepatoma tissue culture cells. Rapid reversible inactivation of soluble enzyme.

Concanavalin A added to intact cells at 37 degrees caused rapid and reversible inactivation of a soluble enzyme, tyrosine aminotransferase, in two lines of rat hepatoma tissue culture cells grown in monolayer culture. This temperature-dependent process was independent of de novo protein and RNA synthesis and independent of increased uptake of Ca2+ and Mg2+ or glucose. The inactivation could be reversed by adding alpha-methyl-D-mannopyranoside a competing sugar for concanavalin A binding. Other lectins known to bind to different sugars did not bring about the inactivation of tyrosine aminotransferase. Addition of concanavalin A did not result in the inactivation of another soluble enzyme, lactic dehydrogenase. The maintenance of tyrosine aminotransferase in an inactive form after the binding of concanavalin A to the cells required the continued presence of concanavalin A. This effect of concanavalin A could not be mimicked either by dibutyryl cyclic adenosine or guanosine monophosphoric acid. Incubation of cell extracts with concanavalin A did not result in inactivation nor did mixing of extracts from concanavalin A-treated cells with extracts from untreated cells. On the basis of these results we conclude that the following are the essential requirements for concanavalin A to bring about the inactivation of tyrosine aminotransferase: (a) the binding of native concanavalin A to the cells; (b) integrity of certain structural elements of the cells.     

Protection of tyrosine aminotransferase against proteolytic digestion by nucleotide derivatives

The abilities of several nucleotides to protect tyrosine aminotransferase (L-tyrosine: 2-oxoglutarate aminotransferase, EC 2.6.1.5) against proteolytic inactivation in vitro have been examined as part of an ongoing investigation of the role of cyclic GMP in the intracellular degradation of the hepatic enzyme. Although neither cyclic GMP nor cyclic AMP was found to exert such a protective effect, certain nucleotide analogs were observed to inhibit the inactivation of tyrosine aminotransferase by trypsin and chymotrypsin. The nucleotides which conferred the strongest protection were the dibutyryl derivatives of cyclic GMP and cyclic AMP. This phenomenon appears to require a purine nucleotide with hydrophobic substituent(s), while the cyclic phosphate is not essential. The nucleotides probably act by direct interaction with tyrosine aminotransferase as indicated by changes in kinetic properties and heat stability of the enzyme and by their failure to inhibit trypsin when other protein substrates, including another aminotransferase, were used. Dibutyryl cyclic AMP was shown to block the appearance of a characteristic 43 kDa tryptic cleavage product of tyrosine aminotransferase but not the conversion of the native 54 kDa form to a size of 50 kDa. Arguments are presented against the involvement of the protective effect in the actions of dibutyryl cyclic nucleotides on tyrosine aminotransferase in cells.     

Aminotransferases for aromatic amino acids and aspartate in Bacillus subtilis.

Two proteins (form A and form B2) with aromatic-amino-acid aminotransferase activity were detected in extracts of Bacillus subtilis. A histidinol phosphate aminotransferase (protein B1) with aminotransferase activity for the aromatic amino acids was also present. The aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) (protein C) also displayed similar activity. Each of the four proteins was isolated free from the others by the successive application of DEAE-cellulose column chromatography and flat-bed isoelectric focusing at pH range 4-6. Form B2 is the major form of the aromatic-amino-acid aminotransferase (aromatic-amino-acid:2-oxoglutarate amino-transferase, EC 2.6.1.57) and the Km values of tyrosine and phenylalanine with this form are somewhat lower than with the minor form A. The Km of tyrosine with histidinol phosphate aminotransferase (protein B1) is in the same range, but the Km of phenylalanine with this enzyme is 12-20 times higher than the corresponding values with the two forms of the aromatic-amino-acid amino-transferase. Apparent molecular weights were estimated with Sephadex gel filtration to be approx. 73 000, 64 000, 54 000 and 66 000 for form A, form B2, histidinol phosphate aminotransferase and aspartate aminotransferase, respectively. Form B2 is being reported for the first time in this communication.     

Functional carboxyl groups in the red cell anion exchange protein. Modification with an impermeant carbodiimide

Anion exchange in human red blood cell membranes was inactivated using the impermeant carbodiimide 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)-carbodiimide (EAC). The inactivation time course was biphasic: at 30 mM EAC, approximately 50% of the exchange capacity was inactivated within approximately 15 min; this was followed by a phase in which irreversible exchange inactivation was approximately 100-fold slower. The rate and extent of inactivation was enhanced in the presence of the nucleophile tyrosine ethyl ester (TEE), suggesting that the inactivation is the result of carboxyl group modification. Inactivation (to a maximum of 10% residual exchange activity) was also enhanced by the reversible inhibitor of anion exchange 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) at concentrations that were 10(3)-10(4) times higher than those necessary for inhibition of anion exchange. The extracellular binding site for stilbenedisulfonates is essentially intact after carbodiimide modification: the irreversible inhibitor of anion exchange 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) eliminated (most of) the residual exchange activity: DNDS inhibited the residual (DIDS-sensitive) Cl- at concentrations similar to those that inhibit Cl- exchange of unmodified membranes: and Cl- efflux is activated by extracellular Cl-, with half-maximal activation at approximately 3 mM Cl-, which is similar to the value for unmodified membranes. But the residual anion exchange function after maximum inactivation is insensitive to changes of extra- and intracellular pH between pH 5 and 7. The titratable group with a pKa of approximately 5.4, which must be deprotonated for normal function of the native anion exchanger, thus appears to be lost after EAC modification.     

The relative stability of liver cytosol enzymes incubated in vitro

1. Relative rates of enzyme inactivation were measured in liver slices, homogenates and cytosol fractions as well as in the presence of trypsin and at acid pH. The enzymes chosen are all present in the cytosol fraction of rat liver, and have widely different degradation rate constants in vivo. 2. The inactivation rates of lactate dehydrogenase, fructose bisphosphate aldolase, glucose 6-phosphate dehydrogenase, glucokinase, phosphoenolpyruvate carboxykinase (GTP), l-serine dehydratase and thymidine kinase in liver preparations at neutral pH are in a similar order to the rate constants of degradation of these enzymes in the intact animal. 3. The two exceptions of this general correlation were tyrosine aminotransferase, which was stable in vitro but not in vivo, and glyceraldehyde phosphate dehydrogenase, which shows the reverse pattern. 4. These findings generally support the concept that the same factors are responsible for enzyme inactivation in vitro as occur in the intact tissue.     

Amino acid residues essential for catalysis by peptidyl dipeptidase-4 from Pseudomonas maltophilia

To assess residues essential for catalysis by prokaryotic peptidyl dipeptidase-4, the enzyme was subjected to chemical modification by a series of reagents. Treatment with either tetranitromethane or N-acetylimidazole abolished catalytic activity. Hydroxylamine reversed inactivation by acetylimidazole only. Thus, an essential tyrosine is indicated. Enzymatic activity also was quenched by either trinitrobenzenesulfonic acid or diethyl pyrocarbonate. Inactivation by these reagents was not reversed by hydroxylamine. These data suggest an essential lysine. The competitive inhibitor Phe-Arg protected partially against inactivation by tetranitromethane, and fully against inactivation by N-acetylimidazole. The substrate Hip-Phe-Arg protected against inactivation by trinitrobenzenesulfonic acid and diethyl pyrocarbonate. Thus, both tyrosine and lysine are located at the catalytic site.     

pH-dependent modulation of Kv1.3 inactivation: role of His399

The Kv1.3 K⁺ channel lacks N-type inactivation, but during prolonged depolarized periods it inactivates via the slow (P/C type) mechanism. It bears a titratable histidine residue in position 399 (equivalent of Shaker 449), a site known to influence the rate of slow inactivation. As opposed to several other voltage-gated K⁺ channels, slow inactivation of Kv1.3 is slowed when extracellular pH (pH_o) is lowered under physiological conditions. Our findings are as follows. First, when His399 was mutated to a lysine, arginine, leucine, valine or tyrosine, extracellular acidification (pH 5.5) accelerated inactivation reminiscent of other Kv channels. Second, inactivation of the wild-type channel was accelerated by low pH_o when the ionic strength of the external solution was raised. Inactivation of the H399K mutant was also accelerated by high ionic strength at pH 7.35 but not the inactivation of H399L. Third, after the external application of blocking barium ions, recovery of the wild-type current during washout was slower in low pH_o. Fourth, the dissociation rate of Ba²⁺ was pH insensitive for both H399K and H399L. Furthermore, Ba²⁺ dissociation rates were equal for H399K and the wild type at pH 5.5 and were equal for H399L and the wild type at pH 7.35. These observations support a model in which the electric field of the protonated histidines creates a potential barrier for potassium ions just outside the external mouth of the pore that hinders their exit from the binding site controlling inactivation. In Kv1.3, this effect overrides the generally observed speeding of slow inactivation when pH_o is reduced. extracellular pH; potassium channel; histidine; barium; high ionic strength     

A pathway for the interconversion of hexose and pentose in the parasitic amoeba Entamoeba histolytica.

The following three potent inhibitors of hepatocytic proteolysis were investigated to see if they would inhibit the intracellular inactivation of enzymes: chymostatin and leupeptin (proteinase inhibitors) and methylamine (a lysosomotropic weak base). Chymostatin inhibited the inactivation of two of the three enzymes tested: tyrosine aminotransferase (EC 2.6.1.5) and tryptophan oxygenase (tryptophan 2,3-dioxygenase, EC 1.13.11.11). Leupeptin had no effect on any of the enzymes, whereas methylamine had only a weak inhibitory effect on tyrosine aminotransferase inactivation. Apparently proteolytic cleavage (probably by a non-lysosomal proteinase, since only chymostatin is effective) is involved in the inactivation of tyrosine aminotransferase and tryptophan oxygenase. The third enzyme, benzopyrene hydroxylase (flavoprotein-linked mono-oxygenase, EC 1.14.14.1), is probably inactivated by a non-proteolytic mechanism.     

Cross-linked stabilization of Escherichia coli penicillin G acylase against pH by dextran-dialdehyde polymers

The inactivation kinetics of Escherichia coli penicillin G acylase (PGA), and cross-linked stabilization of the enzyme by dextran-dialdehyde derivatives of molecular weights of 11500, 37000 and 71000, were similar from pH 2 to pH 10. Inactivation of the native and modified PGA obeyed first order kinetics. The lowest inactivation rate constants for native and dextran-11500-dialdehyde modified PGA were 9.0310 and 1.5310 min respectively at pH 7.0. The highest pH stabilization (nearly ten-fold) was obtained at pH 7.0.