Mapping of reactive sulfhydryls in avian liver 3-hydroxy-3-methylglutaryl coenzyme A synthase

Avian liver mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A synthase contains seven sulfhydryls per 53 kDa subunit. Peptides that harbor these sulfhydryls can be mapped by reverse-phase HPLC separation of tryptic digests of denatured 14C-carboxymethylated enzyme. Native enzyme is inactivated by a variety of reagents that target cysteine residues. Of particular interest is the enzyme's sensitivity to reagents (e.g., CdCl2, copper phenanthroline) that target vicinal thiols. The identity of the cysteines which are modified by these reagents can be determined by peptide mapping after denaturation. 14C-carboxymethylation and trypsin digestion of the sample. While the extent of reaction of any particular cysteinyl sulfhydryl depends on the identity of the reagent employed, three of the protein's seven cysteinyl sulfhydryls are frequently modified upon inactivation of the enzyme. The peptides which contain these reactive sulfhydryls have been isolated and their sequences have been determined by Edman degradation techniques. Comparison of these sequences with the deduced primary structure of the rodent cytosolic enzyme (Gil et al. (1986) J. Biol. Chem. 261, 3710) indicates strong homologies. These homologies allow assignment of the reactive residues as Cys-129, Cys-224 and Cys-268. The sensitivity of these residues to reagents that target vicinal thiols, coupled with the fact that cys-129 is the residue involved in formation of the acyl-S-enzyme intermediate (Vollmer et al. (1988) Biochemistry 27, 4288), suggests that these three residues may be closely juxtaposed within the enzyme's catalytic domain.     

Disulfide-bond formation in the H+-pyrophosphatase of Streptomyces coelicolor and its implications for redox control and enzyme structure

Redox control of disulfide-bond formation in the H+-pyrophosphatase of Streptomyces coelicolor was investigated using cysteine mutants expressed in Escherichia coli. The wild-type enzyme, but not a cysteine-less mutant, was reversibly inactivated by oxidation. To determine the residues involved in oxidative inactivation, different cysteine residues were replaced. Analysis with a cysteine-modifying reagent revealed that the formation of a disulfide bond between cysteines 253 and 621 was responsible for enzyme inactivation. This result suggests that residues in different cytoplasmic loops are close to each other in the tertiary structure. Both cysteine residues are conserved in K+-independent (type II) H+-pyrophosphatases.     

Inhibition of cathepsins and related proteases by amino acid, peptide, and protein hydroperoxides

Reaction of radicals in the presence of O2, and singlet oxygen, with some amino acids, peptides, and proteins yields hydroperoxides. These species are key intermediates in chain reactions and protein damage. Previously we have shown that peptide and protein hydroperoxides react rapidly with thiols, and that this can result in inactivation of thiol-dependent enzymes. The major route for the cellular removal of damaged proteins is via catabolism mediated by proteosomal and lysosomal pathways; cysteine proteases (cathepsins) play a key role in the latter system. We hypothesized that inactivation of cysteine proteases by hydroperoxide-containing oxidised proteins may contribute to the accumulation of modified proteins within cells. We show here that thiol-dependent cathepsins, either isolated or in cell lysates, are rapidly and efficiently inactivated by amino acid, peptide, and protein hydroperoxides in a time- and concentration-dependent manner; this occurs with similar efficacy to equimolar H2O2. Inactivation involves reaction of the hydroperoxide with Cys residues as evidenced by thiol loss and formation of sulfenic acid intermediates. Structurally related, non-thiol-dependent cathepsins are less readily inactivated by these hydroperoxides. This inhibition, by oxidized proteins, of the system designed to remove modified proteins, may contribute to the accumulation of damaged proteins in cells subject to oxidative stress.     

Endogenous ligands of rat lung beta-galactoside-binding protein (galaptin) isolated by affinity chromatography on carboxyamidomethylated-galaptin-Sepharose.

Rat lung beta-galactoside-binding protein (galaptin) is developmentally regulated during postnatal lung development. In common with other vertebrate galaptins, it is very labile when purified and dependent on the presence of exogenous thiol reagents. Reaction of rat lung galaptin with iodoacetamide resulted in a stable active carboxyamidomethylated galaptin that could be coupled to Sepharose. The resultant affinity matrix bound asialoglycoproteins, and these could be quantitatively eluted with disaccharide haptens. The carboxyamidomethylated-galaptin-Sepharose affinity matrix was used to search for endogenous ligands in 13-day-rat lung. Cytosolic fractions of developing rat lung contained no moieties that could be specifically eluted with disaccharide hapten. Only when membranous fractions were extracted with 1% Triton were glycoproteins solubilized that bound to the affinity matrix and could be specifically eluted with disaccharide hapten. The eluted glycoproteins were potent inhibitors of galaptin binding to asialo-orosomucoid. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis identified these glycoproteins as being of high Mr, with three components of Mr 160000-200000 and a smaller component of Mr 75000. This is the first evidence for specific membrane-associated glycoproteins being the ligands of rat lung galaptin.     

Studies on the reactivity of the essential cysteine of thymidylate synthetase

The reaction of one of the four cysteinyl residues of thymidylate synthetase from methotrexate-resistant Lactobacillus casei with a variety of sulfhydryl reagents results in complete inhibition of the enzyme. Kinetic studies indicate that the rates of reactivity of the reagents tested are N-ethylmaleimide > iodoacetamide > N-(iodoacetylaminoethyl)-S-naphthylamine-1-sulfonic acid > iodoacetic acid. The enzyme is also inactivated by 5-Hg-deoxyuridylate, a compound which reacts stoichiometrically with a single cysteine. Unlike the other reagents, the inhibition produced by this compound can be completely reversed by added thiols. The same cysteine appears to react with all of the sulfhydryl reagents, as shown by competition experiments and by protection against inactivation by deoxyuridylate. Even at a 100-fold excess of the alkylating agents, only one of the four cysteines in the native enzyme was reactive, attesting to the uniqueness of this residue. Carboxypeptidase A inactivation of the enzyme does not affect either the binding of deoxyuridylate to the enzyme or the reactivity of N-ethylmaleimide with the “catalytic” cysteine. Under denaturing conditions, all four cysteinyl residues react with N-ethylmaleimide or iodoacetate, as shown by identifying the reaction products by amino acid analysis. The covalent ternary complex [(+)5,10-methylenetetrahydrofolate-5-fluorodeoxyuridylate-thymidylate synthetase] (molar ratio = 2:2:1) revealed only two cysteinyl residues capable of reacting with N-ethylmaleimide or iodoacetate upon denaturation. From these data, it appears that one cysteine is involved in the binding of deoxyuridylate and that two of the enzyme's four cysteines are responsible for binding 5-fluorodeoxyuridylate in the ternary complex.     

Redox-regulated cochaperone activity of the human DnaJ homolog Hdj2

The human DnaJ homolog Hdj2 is a cochaperone containing a cysteine-rich zinc finger domain. We identified a specific interaction of Hdj2 with the cellular redox enzyme thioredoxin using a yeast two-hybrid assay and a coimmunoprecipitation assay, thereby investigating how the redox environment of the cell regulates Hdj2 function. In reconstitution experiments with Hsc70, we found that treatment with H2O2 caused the oxidative inactivation of Hdj2 cochaperone activity. Hdj2 inactivation paralleled the oxidation of cysteine thiols and concomitant release of coordinated zinc, suggesting a role of cysteine residues in the zinc finger domain of Hdj2 as a redox sensor of chaperone-mediated protein-folding machinery. H2O2-induced negative regulation of Hdj2 cochaperone activity was also confirmed in mammalian cells using luciferase as a foreign reporter cotransfected with Hsc70 and Hdj2. The in vivo oxidation of cysteine residues in Hdj2 was detected only in thioredoxin-knockdown cells, implying that thioredoxin is involved in the in vivo reduction. The oxidative inactivation of Hdj2 was reversible. Wild-type thioredoxin notably recovered the oxidatively inactivated Hdj2 activity accompanied by the reincorporation of zinc, whereas the catalytically inactive mutant thioredoxin (Cys32Ser/Cys35Ser) did not. Taken together, we propose that oxidation and reduction reversibly regulate Hdj2 function in response to the redox states of the cell.     

Formation of an intramolecular cystine disulfide during the reaction of 8-azidoguanosine 5'-triphosphate with cytosolic phosphoenolpyruvate carboxykinase (GTP) causes inactivation without photolabeling

Phosphoenolpyruvate carboxykinase (GTP) (PEPCK) specifically utilizes a guanosine or inosine nucleotide as a substrate, yet it does not share extended sequence homology with other GTP-binding proteins, and the molecular basis for its nucleotide specificity is not understood. In an effort to locate the enzyme's nucleotide-binding site, we have studied the interaction of cytosolic PEPCK from rat liver with the photoprobe 8-azidoGTP, which fulfills the criteria of a specific photoaffinity label for PEPCK. The photoprobe binds reversibly to the enzyme prior to modification and at low concentrations causes greater than 60% inactivation (Ki = 1.2 microM). GTP provides nearly complete protection against inactivation by 8-azidoGTP, whereas phosphoenolpyruvate and metal ions provide partial protection. In addition, the photoprobe is a substrate for the enzyme and has a Km similar to that for GTP. However, the extent of covalent modification by [32P]8-azidoGTP as measured by three independent techniques is significantly lower than the extent of enzyme inactivation. Further investigation of this anomaly has revealed that the loss in enzymatic activity is caused by modification of a critical cysteine residue in a reaction that does not terminate with covalent attachment of the photolabel. Quantitation of the total free thiols of modified PEPCK shows that 2 mol of cysteine is lost per mole of inactivated enzyme. These results indicate that the photoinactivation of PEPCK by 8-azidoGTP is caused by the formation of an intramolecular cystine disulfide bridge, thus providing evidence for the existence of a pair of proximal cysteine residues within the GTP-binding site. The interaction of cysteine residues with the reactive photogenerated derivatives of 8-azidopurines is discussed.     

An essential cysteine residue located in the vicinity of the FAD-binding site in short-chain, medium-chain, and long-chain acyl-CoA dehydrogenases from rat liver mitochondria

Medium-chain and long-chain acyl-CoA dehydrogenases from rat liver have been purified in two forms, holoenzymes containing FAD and apoenzymes which do not contain this cofactor. In contrast, short-chain acyl-CoA dehydrogenase can only be isolated as the holoenzyme. Marked differences in the reactivity to organic sulfhydryl reagents were observed between the apo and holo forms of these enzymes. While the two apoenzymes were severely inactivated by N-ethylmaleimide (NEM), p-chloromercuribenzoate (pCMB), and iodoacetate (IAA), the two corresponding holoenzymes were not susceptible to these reagents. The inactivation of the two apoenzymes by NEM followed pseudo-first order kinetics. Incubation of the apoenzymes with FAD completely prevented the inactivation by the organic sulfhydryl reagents. Methylmercury halides (iodide or chloride) inactivated both the apo and holo forms of medium-chain and long-chain acyl-CoA dehydrogenases. On the other hand, holo-short-chain acyl-CoA dehydrogenase behaved somewhat differently from the other two holoenzymes in that it was inactivated by pCMB (but not NEM or IAA) following a pseudo-first order process. The titration of the two apoenzymes with [14C]NEM and that of the holo-short-chain acyl-CoA dehydrogenase with [14C]pCMB indicated that all three acyl-CoA dehydrogenases contain a single essential cysteine residue/subunit. In the inactivation of holo-medium-chain and holo-long-chain acyl-CoA dehydrogenases with methylmercury halide, the same essential cysteine residue was modified without perturbing or releasing the enzyme-bound FAD. The inactivations of the three holoenzymes by appropriate organic sulfhydryl reagents were prevented by prior incubation with substrate. These experimental results indicate that the essential cysteine residue is located in the vicinity of the FAD- and substrate-binding sites within the active center of the enzymes. It appears, however, that this cysteine residue does not participate directly in FAD binding.     

Essential tyrosyl residues in Lactobacillus casei thymidylate synthetase

Sulfhydryl-blocked thymidylate synthetase (EC 2.1.1.4.5) is rapidly inactivated by low concentrations of tetranitromethane. This reagent first nitrates two non-essential tyrosines per dimeric enzyme molecule followeed by two essential tyrosines with no oxidation of sulfhydryl groups. dUMP affords significant protection against inactivation. These results suggest that essential tyrosyl residues are present in the active sites of the enzyme.     

Group-selective reagent modification of the sodium- and chloride-coupled glycine transporter under native and reconstituted conditions.

Glycine transporter from rat brain stem and spinal cord is inactivated by specific sulfhydryl reagents. Modification of lysine residues also promotes a decrease of the transporter activity but in a lesser extent than that promoted by thiol group reagents. Mercurials showed a more marked inhibitory effect than maleimide derivatives. SH groups display a similar reactivity for p-chloromercuribenzenesulfonate (pCMBS) and mersalyl in synaptosomal membrane vesicles and proteoliposomes reconstituted with the solubilized transporter. However, different reactivity is observed with N-ethylmaleimide (MalNEt), the greatest effect being attained in membrane vesicles. The rate of inactivation by pCMBS and MalNEt is pseudo-first-order showing time- and concentration-dependence. pCMBS and MalNEt decrease the Vmax for glycine transport and to a lesser extent act on the apparent Km. Treatment with dithiothreitol (DTT) of the transporter modified by pCMBS results in a complete restoration of transporter activity indicating that the effect exercised by the reagent is specific for cysteine residues on the protein. It is concluded that SH groups are involved in the glycine transporter function and that these critical residues are mostly located in a relatively hydrophilic environment of the protein.     

Chemical modifications of alpha1,6-fucosyltransferase define amino acid residues of catalytic importance

alpha1,6-Fucosyltransferase (alpha6FucT) of human platelets was subjected to the action of phenylglyoxal (PLG), pyridoxal-5'-phosphate/NaBH(4) (PLP), and diethyl pyrocarbonate (DEPC) the reagents that selectively modify the structure of amino acids arginine, lysine and histidine, respectively, as well as to N-ethylmaleimide (NEM), mersalyl, p-chloromercuribenzoate (pCMB), iodoacetate, iodoacetamide, and methyl iodide that react with sulfhydryl group of cysteine. In addition, we treated the enzyme with beta-mercaptoethanol, a reagent that disrupts disulfide bonds. All reagents except NEM significantly inactivated alpha6FucT. Protection against the action of PLG, PLP and sulfhydryl modifying reagents was offered by GDP-fucose, GDP, and the acceptor substrate, a transferrin-derived biantennary glycopeptide with terminal GlcNAc residues. Neither donor nor acceptor substrate offered, however, any protection against inactivation by DEPC or beta-mercaptoethanol. We conclude that arginine, cysteine and probably lysine residues are present in, or closely by, the donor and acceptor substrate binding domains of the enzyme, whereas histidine may be a part of its catalytic domain. However, the primary structure of alpha6FucT does not show cysteine residues in proximity to the postulated GDP-fucose-binding site and acceptor substrate binding site of the enzyme that contains two neighboring arginine residues and one lysine residue (Glycobiol. 10 (2000) 503). To rationalize our results we postulate that platelet alpha6FucT is folded through disulfide bonds that bring together donor/acceptor-binding- and cysteine- and lysine-rich, presumably acceptor substrate binding sites, thus creating a catalytic center of the enzyme.     

Specific and reversible inactivation of Phycomyces blakesleeanus isocitrate lyase by ascorbate-iron: role of two redox-active cysteines

Phycomyces blakesleeanus isocitrate lyase (EC 4.1.3.1) is in vivo reversibly inactivated by hydrogen peroxide. The purified enzyme showed reversible inactivation by an ascorbate plus Fe(2+) system under aerobic conditions. Inactivation requires hydrogen peroxide; was prevented by catalase, EDTA, Mg(2+), isocitrate, GSH, DTT, or cysteine; and was reversed by thiols. The ascorbate served as a source of hydrogen peroxide and also reduced the Fe(3+) ions produced in a "site-specific" Fenton reaction. Two redox-active cysteine residues per enzyme subunit are targets of oxidative modification; one of them is located at the catalytic site and the other at the metal regulatory site. The oxidized enzyme showed covalent and conformational changes that led to inactivation, decreased thermal stability, and also increased inactivation by trypsin. These results represent an example of redox regulation of an enzymatic activity, which may play a role as a sensor of redox cellular status.     

Highly reactive cysteine residues are part of the substrate binding site of mammalian dipeptidyl peptidases III

Dipeptidyl peptidase III (DPP III) is a cytosolic zinc-exopeptidase involved in the intracellular protein catabolism of eukaryotes. Although inhibition by thiol reagents is a general feature of DPP III originating from various species, the function of activity important sulfhydryl groups is still inadequately understood. The present study of the reactivity of these groups was undertaken in order to clarify their biological significance.The inactivation kinetics of human and rat DPP III by sulfhydryl reagent p-hydroxy-mercuribenzoate (pHMB) was monitored by determination of the enzyme's residual activity with fluorimetric detection.Inactivation of this human enzyme exhibited pseudo-first-order kinetics, suggesting that all reactive SH-groups have equivalent reactivity, and the second-order rate constant was calculated to be 3523+/-567M(-1)min(-1). Rat DPP III was hyperreactive to pHMB and showed biphasic kinetics indicating two classes of reactive SH-groups. The second-order rate constants of 3540M(-1)s(-1) for slower reacting sulfhydryl, and 21,855M(-1)s(-1) for faster reacting sulfhydryl were obtained from slopes of linear plots of pseudo-first-order constants versus reagent concentration. Peptide substrates protected both mammalian DPPs III from inactivation by pHMB. Physiological concentrations of biological thiols and H(2)O(2) inactivated the rat DPP III. Human enzyme was resistant to H(2)O(2) attack and less affected by reduced glutathione (GSH) than the rat homologue. A significantly lower DPP III level, determined by activity measurement and Western blotting, was found in the cytosols of highly oxygenated rat tissues.These results provide kinetic evidence that cysteine residues are involved in substrate binding of mammalian DPPs III.     

Selenium-containing amino acids are targets for myeloperoxidase-derived hypothiocyanous acid: determination of absolute rate constants and implications for biological damage

Elevated MPO (myeloperoxidase) levels are associated with multiple human inflammatory pathologies. MPO catalyses the oxidation of Cl-, Br- and SCN- by H2O2 to generate the powerful oxidants hypochlorous acid (HOCl), hypobromous acid (HOBr) and hypothiocyanous acid (HOSCN) respectively. These species are antibacterial agents, but misplaced or excessive production is implicated in tissue damage at sites of inflammation. Unlike HOCl and HOBr, which react with multiple targets, HOSCN targets cysteine residues with considerable selectivity. In the light of this reactivity, we hypothesized that Sec (selenocysteine) residues should also be rapidly oxidized by HOSCN, as selenium atoms are better nucleophiles than sulfur. Such oxidation might inactivate critical Sec-containing cellular protective enzymes such as GPx (glutathione peroxidase) and TrxR (thioredoxin reductase). Stopped-flow kinetic studies indicate that seleno-compounds react rapidly with HOSCN with rate constants, k, in the range 2.8×10(3)-5.8×10(6) M-1·s-1 (for selenomethionine and selenocystamine respectively). These values are ~6000-fold higher than the corresponding values for H2O2, and are also considerably larger than for the reaction of HOSCN with thiols (16-fold for cysteine and 80-fold for selenocystamine). Enzyme studies indicate that GPx and TrxR, but not glutathione reductase, are inactivated by HOSCN in a concentration-dependent manner; k for GPx has been determined as ~5×105 M-1·s-1. Decomposed HOSCN did not induce inactivation. These data indicate that selenocysteine residues are oxidized rapidly by HOSCN, with this resulting in the inhibition of the critical intracellular Sec-dependent protective enzymes GPx and TrxR.     

Molecular basis of superreactivity of cysteine residues 31 and 32 of seminal ribonuclease

The molecular basis of the high reactivity toward reducing agents of intersubunit disulfides at positions 31 and 32 of dimeric bovine seminal ribonuclease was investigated by studying in the monomeric enzyme the fast reaction kinetics with disulfides of the adjacent cysteine-31 and -32, exposed by selective reduction of the intersubunit disulfides. Negatively charged and neutral disulfide reagents were used for measuring the thiol reaction rates at neutral pH. The kinetics studied as a function of pH permitted us to define pK values for the thiols of interest and indicated the possibility of determining pK values of SH groups in proteins indirectly by measuring the kinetics of reactivity of the SH groups with a disulfide reagent. The results were compared with those obtained under identical conditions with synthetic thiol peptides and model compounds. The data indicate that the superreactivity of intersubunit disulfides of seminal ribonuclease is matched by the high reactivity at neutral pH of adjacent cysteine residues 31 and 32, as compared to all small thiol compounds tested. The synthetic hexapeptide segment of seminal ribonuclease Ac-Met-Cys-Cys-Arg-Lys-Met-OH, which includes the two cysteine residues of interest, was even more reactive. These data, and the other results reported in this paper, led to the conclusion that the superreactivity at neutral pH of cysteine residues at positions 31 and 32 of bovine seminal ribonuclease is primarily dependent on the nearby presence of positively charged groups, particularly the epsilon-NH2 of lysine-34, and is influenced by the adjacency of the two thiols and by the protein tertiary structure.     

A Redox-resistant Sirtuin-1 Mutant Protects against Hepatic Metabolic and Oxidant Stress

Sirtuin-1 (SirT1), a member of the NAD⁺-dependent class III histone deacetylase family, is inactivated in vitro by oxidation of critical cysteine thiols. In a model of metabolic syndrome, SirT1 activation attenuated apoptosis of hepatocytes and improved liver function including lipid metabolism. We show in SirT1-overexpressing HepG2 cells that oxidants (nitrosocysteine and hydrogen peroxide) or metabolic stress (high palmitate and high glucose) inactivated SirT1 by reversible oxidative post-translational modifications (OPTMs) on three cysteines. Mutating these oxidation-sensitive cysteines to serine preserved SirT1 activity and abolished reversible OPTMs. Overexpressed mutant SirT1 maintained deacetylase activity and attenuated proapoptotic signaling, whereas overexpressed wild type SirT1 was less protective in metabolically or oxidant-stressed cells. To prove that OPTMs of SirT1 are glutathione (GSH) adducts, glutaredoxin-1 was overexpressed to remove this modification. Glutaredoxin-1 overexpression maintained endogenous SirT1 activity and prevented proapoptotic signaling in metabolically stressed HepG2 cells. The in vivo significance of oxidative inactivation of SirT1 was investigated in livers of high fat diet-fed C57/B6J mice. SirT1 deacetylase activity was decreased in the absence of changes in SirT1 expression and associated with a marked increase in OPTMs. These results indicate that glutathione adducts on specific SirT1 thiols may be responsible for dysfunctional SirT1 associated with liver disease in metabolic syndrome.     

Inactivation of porcine kidney betaine aldehyde dehydrogenase by hydrogen peroxide

Concentrated urine formation in the kidney is accompanied by conditions that favor the accumulation of reactive oxygen species (ROS). Under hyperosmotic conditions, medulla cells accumulate glycine betaine, which is an osmolyte synthesized by betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8). All BADHs identified to date have a highly reactive cysteine residue at the active site, and this cysteine is susceptible to oxidation by hydrogen peroxide. Porcine kidney BADH incubated with H(2)O(2) (0-500 μM) lost 25% of its activity. However, pkBADH inactivation by hydrogen peroxide was limited, even after 120 min of incubation. The presence of coenzyme NAD(+) (10-50 μM) increased the extent of inactivation (60%) at 120 min of reaction, but the ligands betaine aldehyde (50 and 500 μM) and glycine betaine (100 mM) did not change the rate or extent of inactivation as compared to the reaction without ligand. 2-Mercaptoethanol and dithiothreitol, but not reduced glutathione, were able to restore enzyme activity. Mass spectrometry analysis of hydrogen peroxide inactivated BADH revealed oxidation of M278, M243, M241 and H335 in the absence and oxidation of M94, M327 and M278 in the presence of NAD(+). Molecular modeling of BADH revealed that the oxidized methionine and histidine residues are near the NAD(+) binding site. In the presence of the coenzyme, these oxidized residues are proximal to the betaine aldehyde binding site. None of the oxidized amino acid residues participates directly in catalysis. We suggest that pkBADH inactivation by hydrogen peroxide occurs via disulfide bond formation between vicinal catalytic cysteines (C288 and C289).     

The effect of mixed-function oxidation of enzymes on their susceptibility to degradation by a nonlysosomal cysteine proteinase

Mixed-function oxidation of Escherichia coli glutamine synthetase by ascorbate, oxygen, and iron has previously been shown to cause inactivation of the enzyme and enhanced susceptibility to proteolytic attack by a variety of proteases. One of these proteases, from rat liver, is a high molecular weight cysteine proteinase which does not degrade native glutamine synthetase at neutral pH. Although inactive, the oxidized glutamine synthetase preparations used in this study were only partially degraded by this proteinase. Some of the subunits were degraded to acid soluble products with no detectable intermediates; the remaining subunits had not become susceptible to proteolytic attack during the limited exposure to the ascorbate mixed-function oxidation system. Several mammalian enzymes which are known to be inactivated by mixed-function oxidation were tested as substrates for the proteinase. Native rabbit muscle enolase and pyruvate kinase were resistant to degradation, but their oxidatively inactivated forms were degraded. Oxidized phosphoglycerate kinase and creatine kinase were also preferentially degraded. Moreover, trypsin degraded oxidized preparations of all of these enzymes faster than control preparations. Oxidative inactivation of superoxide dismutase by hydrogen peroxide caused a slight increase in susceptibility to proteolytic attack, but the enzyme was still relatively resistant to degradation both by the cysteine proteinase and by trypsin. Although oxidation conditions may not have been optimal for demonstrating enhanced proteolytic susceptibility, the results do indicate that mixed-function oxidation can render some mammalian enzymes, as well as bacterial glutamine synthetase, susceptible to degradation. Mixed-function oxidation of these proteins may be a mechanism of marking them for intracellular turnover.     

Rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Identification of essential sulfhydryl residues in the primary sequence of the enzyme

The kinase and sugar phosphate exchange reactions of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase were inactivated by treatment with 5'-p-fluorosulfonylbenzoyladenosine or 8-azido-ATP, but activity could be restored by the addition of dithiothreitol. This inactivation was accompanied by incorporation of 5'-p-sulfonylbenzoyl[8-14C]adenosine into the enzyme that was not released by the addition of dithiothreitol. The lack of effect of ATP analogs on the ATP/ADP exchange or on bisphosphatase activity and reversal of their effects on the kinase and sugar phosphate reactions by dithiothreitol suggest that 1) they reacted with sulfhydryl groups important for sugar phosphate binding in the kinase reaction, and 2) the inactivation of the kinase by these analogs involves a specific reaction that is not related to their general mechanism of attacking nucleotide-binding sites. In addition, alkylation of the enzymes' sulfhydryls with iodoacetamide prevented inactivation by 5'-p-fluorosulfonylbenzoyladenosine, suggesting that the same thiols were involved. o-Iodosobenzoate inactivated the kinase and sugar phosphate exchange; the inactivation was reversed by dithiothreitol; but there was no effect on the bisphosphatase or nucleotide exchange, indicating that oxidation occurred at the same sulfhydryl that are associated with sugar phosphate binding. ATP or ADP, but not fructose 6-phosphate, protected these groups from modification by 5'-p-fluorosulfonylbenzoyladenosine, 8-azido-ATP, and o-iodosobenzoate. ATP also induced dramatic changes in the circular dichroism spectrum of the enzyme, suggesting that adenine nucleotide protection of thiol groups resulted from changes in enzyme secondary structure. Analysis of cyanogen bromide fragments of 14C-carboxamidomethylated enzyme showed that all radioactivity was associated with cysteinyl residues in a single cyanogen bromide fragment. Three of these cysteinyl residues are clustered in a 38-residue region, which probably plays a role in maintaining the conformation of the kinase sugar phosphate-binding site.