Adjacent cysteine residues as a redox switch.

Oxidation of adjacent cysteine residues into a cystine forms a strained eight-membered ring. This motif was tested as the basis for an enzyme with an artificial redox switch. Adjacent cysteine residues were introduced into two different structural contexts in ribonuclease A (RNase A) by site-directed mutagenesis to produce the A5C/A6C and S15C/S16C variants. Ala5 and Ala6 are located in an alpha-helix, whereas Ser15 and Ser16 are located in a surface loop. Only A5C/A6C RNase A had the desired property. The catalytic activity of this variant decreases by 70% upon oxidation. The new disulfide bond also decreases the conformational stability of the A5C/A6C variant. Reduction with dithiothreitol restores full enzymatic activity. Thus, the insertion of adjacent cysteine residues in a proper context can be used to modulate enzymatic activity.     

Improved assay sensitivity of an engineered secreted Renilla luciferase.

We have previously reported the construction of a functional Renilla luciferase enzyme secreted by mammalian cells when fused to the signal peptide of human interleukin-2. The presence of three predicted cysteine residues in the amino acid sequence of Renilla luciferase suggested that its secreted form could contain oxidized sulfhydryls, which might impair enzyme activity. In this work, four secreted Renilla luciferase mutants were constructed to investigate this possibility: three luciferase mutants in which a different cysteine residue was replaced by an alanine residue, and one luciferase mutant in which all three cysteine residues were replaced by alanine residues. Simian cells were transfected with the genes encoding these mutant luciferases, as well as with the original gene construct, and cell culture media were assayed for bioluminescence activity. Only media containing a mutated luciferase with a cysteine to alanine substitution at position 152 in the preprotein showed a marked increase in bioluminescence activity when compared to media containing the original secreted Renilla luciferase. This increase in light emission was due in part to enhanced stability of the mutant enzyme. This new enzyme represents a significant improvement in the sensitivity of the secreted Renilla luciferase assay for monitoring gene expression.     

Role of cysteine residues in Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-CoA reductase. Site-directed mutagenesis and characterization of the mutant enzymes

Each of the four identical subunits of Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase contains two cysteine residues, Cys156 and Cys296 (Beach, M. J., and Rodwell, V. W. (1989) J. Bacteriol. 171, 2994-3001). Both are accessible to modification by sulfhydryl reagents under nondenaturing conditions (Jordan-Starck, T. C., and Rodwell, V. W. (1989) J. Biol. Chem. 264, 17913-17918). We used site-directed mutagenesis to construct three mutant enzymes in which alanine replaced either or both cysteine residues. Mutant enzymes C156A, C296A, and C156/296A were over-expressed in Escherichia coli and were found to be fully active. Following their purification, all four forms of the enzyme were compared with respect to their catalytic efficiency, their affinities for the substrates of all four catalyzed reactions, and for their sensitivity to inactivation by sulfhydryl reagents. Replacement of cysteine residues with alanine residues had no major effect on either the specific activity or the affinity of the enzymes for any substrate. The mutants catalyzed all four HMG-CoA reductase reactions as efficiently as did the wild-type enzyme, and coenzyme A stimulated mevaldehyde reduction to the same extent as for wild-type HMG-CoA reductase. Mutant C156A and the cysteine-free mutant C156/296A were not inactivated by 5,5'-dithiobis(2-nitrobenzoate). By contrast, mutant C296A was inactivated to the same extent as was the wild-type enzyme. Following treatment of the mutant enzymes with N-ethylmaleimide, the four reductase reactions catalyzed by mutant C296A were inactivated to the same extent as for the wild-type enzyme. Neither mutant C156A nor C156/296A was affected by this reagent. We conclude that the sulfhydryl reagent-reactive group whose derivatization leads to loss of enzymatic activity is Cys156. However, this residue is not an essential active site residue since neither substrate binding nor catalysis was affected when it was replaced by alanine. Possible roles of cysteine in maintaining structural stability are discussed.     

Redox regulation of enzymatic activity and proteolytic susceptibility of ribulose-1,5-bisphosphate carboxylase/oxygenase fromEuglena gracilis

The activity of ribulose-1,5-bisphosphate carboxylase/oxygenase fromEuglena gracilis decays steadily when exposed to agents that induce oxidative modification of cysteine residues (Cu²⁺, benzofuroxan, disulfides, arsenite, oxidized ascorbate). Inactivation takes place with a concomitant loss of cysteine sulfhydryl groups and dimerization of large subunits of the enzyme. 40% activity loss induced by the vicinal thiol-reagent arsenite is caused by modification of a few neighbor residues while the almost complete inactivation achieved with disulfides is due to extensive oxidation leading to formation of mixed disulfides with critical cysteines of the protein. In most cases oxidative inactivation is also accompanied by an increased sensitivity to proteolysis by trypsin, chymotrypsin or proteinase K. Both enzymatic activity and resistance to proteolysis can be restored through treatment with several thiols (cysteamine, cysteine, dithiothreitol and, more slowly, reduced glutathione). Redox effectors which are thought to regulate the chloroplast activity (NADPH, ferredoxin and thioredoxin) do not reactivate the oxidized enzyme. When ribulose-1,5-bisphoshate carboxylase/oxygenase is incubated with cystamine/cysteamine mixtures having different disulfide/thiol ratio (r), inactivation takes place around r=1.5 while proteolytic sensitization occurs under more oxidative conditions (r=4). It is suggested that oxidative modification may happen in vivo under exceptional circumstances, such as senescence, bleaching or different kinds of stress, leading to enzyme inactivation and triggering the selective degradation of the carboxylase that has been repeatedly observed during these processes.     

Thermostabilization of recombinant human and bovine CuZn superoxide dismutases by replacement of free cysteines

Human CuZn superoxide dismutase (HSOD) has two free cysteines: a buried cysteine (Cys6) located in a beta-strand, and a solvent accessible cysteine (Cys111) located in a loop region. The highly homologous bovine enzyme (BSOD) has a single buried Cys6 residue. Cys6 residues in HSOD and BSOD were replaced by alanine and Cys111 residues in HSOD by serine. The mutant enzymes were expressed and purified from yeast and had normal specific activities. The relative resistance of the purified proteins to irreversible inactivation of enzymatic activity by heating at 70 degrees C was HSOD Ala6 Ser111 greater than BSOD Ala6 Ser109 greater than BSOD Cys6 Ser109 (wild type) greater than HSOD Ala6 Cys111 greater than HSOD Cys6 Ser111 greater than HSOD Cys111 (wild type). In all cases, removal of a free cysteine residue increased thermostability.     

Early effects of hypoxia/reoxygenation on VEGF,Ang-1, Ang-2 and their receptors in the rat myocardium: Implications for myocardial angiogenesis

The oxidized form of vitamin C (dehydroascorbic acid, DHA) completely and irreversibly inactivates recombinant human hexokinase type I, in a pseudo-first order fashion. The inactivation reaction occurs without saturation, indicating that DHA does not form a reversible complex with hexokinase. Further characterization of this response revealed that the inactivation does not require oxygen and that dithiothreitol, while able to prevent the DHA-mediated loss of enzyme activity, failed to restore the activity of the DHA-inhibited enzyme. Inactivation was not associated with cleavage of the peptide chain or cross-linking. The decay in enzymatic activity was however both dependent on deprotonation of a residue with an alkaline pK_a and associated with covalent binding of DHA to the protein. In addition, inactivation of hexokinase decreased or increased, respectively, in the presence of the substrates glucose or MgATP. Finally, amino acid analysis of the DHA-modified hexokinase revealed a decrease of cysteine residues.Taken together, the above results are consistent with the possibility that covalent binding of the reagent with a thiol group of cysteine is a critical event for the DHA-mediated loss of hexokinase activity.     

Engineered disulfide bonds increase active-site local stability and reduce catalytic activity of a cold-adapted alkaline phosphatase

Alkaline phosphatase is an extracellular enzyme that is membrane-bound in eukaryotes but resides in the periplasmic space of bacteria. It normally carries four cysteine residues that form two disulfide bonds, for instance in the APs of Escherichia coli and vertebrates. An AP variant from a Vibrio sp. has only one cysteine residue. This cysteine is second next to the nucleophilic serine in the active site. We have individually modified seven residues to cysteine that are on two loops predicted to be within a 5 A radius. Four of them formed a disulfide bond to the endogenous cysteine. Thermal stability was monitored by circular dichroism and activity measurements. Global stability was similar to the wild-type enzyme. However, a significant increase in heat-stability was observed for the disulfide-containing variants using activity as a measure, together with a large reduction in catalytic rates (k(cat)) and a general decrease in Km values. The results suggest that a high degree of mobility near the active site and in the helix carrying the endogenous cysteine is essential for full catalytic efficiency in the cold-adapted AP.     

Effect of cysteine residues on the activity of arginyl-tRNA synthetase from Escherichia coli.

Arginyl-tRNA synthetase (ArgRS) from Escherichia coli (E. coli) contains four cysteine residues. In this study, the role of cysteine residues in the enzyme has been investigated by chemical modification and site-directed mutagenesis. Titration of sulfhydryl groups in ArgRS by 5, 5'-dithiobis(2-nitro benzoic acid) (DTNB) suggested that a disulfide bond was not formed in the enzyme and that, in the native condition, two DTNB-sensitive cysteine residues were located on the surface of ArgRS, while the other two were buried inside. Chemical modification of the native enzyme by iodoacetamide (IAA) affected only one DTNB-sensitive cysteine residue and resulted in 50% loss of enzyme activity, while modification by N-ethylmeimide (NEM) affected two DTNB-sensitive residues and caused a complete loss of activity. These results, when combined with substrate protection experiments, suggested that at least the two cysteine residues located on the surface of the molecule were directly involved in substrates binding and catalysis. However, changing Cys to Ala only resulted in slight loss of enzymatic activity and substrate binding, suggesting that these four cysteine residues in E. coli ArgRS were not essential to the enzymatic activity. Moreover, modifications of the mutant enzymes indicated that the two DTNB- and NEM-sensitive residues were Cys(320) and Cys(537) and the IAA-sensitive was Cys(320). Our study suggested that inactivation of E. coli ArgRS by sulfhydryl reagents is a result of steric hindrance in the enzyme.     

Inactivation of chicken mitochondrial phosphoenolpyruvate carboxykinase by o-phthalaldehyde

Chicken liver mitochondrial phosphoenolpyruvate carboxykinase is inactivated by o-phthalaldehyde. The inactivation followed pseudo first-order kinetics, and the second-order rate constant for the inactivation process was 29 M-1 s-1 at pH 7.5 and 25 degrees C. The modified enzyme showed maximal fluorescence at 427 nm upon excitation at 337 nm, consistent with the formation of isoindole derivatives by the cross-linking of proximal cysteine and lysine residues. Activities in the physiologic reaction and in the oxaloacetate decarboxylase reaction were lost in parallel upon modification with o-phthalaldehyde. Plots of (percent of residual activity) versus (mol of isoindole incorporated/mol of enzyme) were biphasic, with the initial loss of enzymatic activity corresponding to the incorporation of one isoindole derivative/enzyme molecule. Complete inactivation of the enzyme was accompanied by the incorporation of 3 mol of isoindole/mol of enzyme. beta-Sulfopyruvate, an isoelectronic analogue of oxaloacetate, completely protected the enzyme from reacting with o-phthalaldehyde. Other substrates provided protection from inactivation, in decreasing order of protection: oxaloacetate greater than phosphoenolpyruvate greater than MgGDP, MgGTP greater than oxalate. Cysteine 31 and lysine 39 have been identified as the rapidly reacting pair in isoindole formation and enzyme inactivation. Lysine 56 and cysteine 60 are also involved in isoindole formation in the completely inactivated enzyme. These reactive cysteine residues do not correspond to the reactive cysteine residue identified in previous iodoacetate labeling studies with the chicken mitochondrial enzyme (Makinen, A. L., and Nowak, T. (1989) J. Biol. Chem. 264, 12148-12157). Protection experiments suggest that the sites of o-phthalaldehyde modification become inaccessible when the oxaloacetate/phosphoenolpyruvate binding site is saturated, and sequence analyses indicate that cysteine 31 is located in the putative phosphoenolpyruvate binding site.     

Oxidative inhibition of human soluble catechol-O-methyltransferase

A common polymorphism in the human gene for catechol-O-methyltransferase results in replacement of Val-108 by Met in the soluble form of the protein (s-COMT) and has been linked to breast cancer and neuropsychiatric disorders. The 108M and 108V variants are reported to differ in their thermal stability, with 108M COMT losing catalytic activity more rapidly. Because human s-COMT contains seven cysteine residues and includes CXXC and CXXS motifs that are associated with thiol-disulfide redox reactions, we examined the effects of reducing and oxidizing conditions on the enzyme. In the absence of a reductant 108M s-COMT lost activity more rapidly than 108V, whereas in the presence of 4 mm dithiothreitol (DTT) we found no significant differences in the stability of the two variants at 37 degrees C. DTT also restored most of the activity that was lost upon incubation at 37 degrees C in the absence of DTT. Mass spectrometry showed that cysteines 188 and 191 formed an intramolecular disulfide bond when s-COMT was incubated with oxidized glutathione, whereas cysteines 69, 95, 157, and 173 formed protein-glutathione adducts. Replacing Cys-95 by serine protected 108M s-COMT against inactivation in the absence of a reductant; C33S and Cys-188 mutations had little effect, and C69S was destabilizing. The sequences surrounding the reactive cysteine residues of human s-COMT and other proteins that form glutathione adducts at identified sites all include Pro and/or Gly and most include a hydrogen-bonding residue, suggesting that glutathiolation at conserved sites plays a physiologically important role.     

Distribution of the Val108/158Met polymorphism of the COMT gene in healthy Mexican population

Catechol-O-methyltransferase (COMT) inactivates the catecholamines adrenaline, noradrenaline and dopamine. On the other hand, some studies have reported that the enzymatic activity of COMT is partly genetically determined. With regard to the COMT gene, the most studied polymorphism is the functional variant Val108/158Met (rs4680), which results in substantial three- to four-fold variations in enzyme activity. To date, the rs4680 polymorphism of COMT has been associated with a number of disorders. In addition, this polymorphism has been found to have important differences in frequency according to the studied population. Therefore, the aim of the present study was to evaluate the frequency of a common single nucleotide polymorphism (SNP) Val108/158Met of the COMT gene in the Mexican population. Accordingly, we recruited 431 healthy volunteers. Our sample consisted of 111 healthy individuals from Mexico City and 320 individuals from the state of Tabasco, Mexico. We observed that Met was the most common allele, ranging from 57% (Tabasco) to 85% (Mexico City). In addition, we analyzed the frequency of Val108/158Met polymorphism of Caucasian (54% Met allele), Asian (29% Met allele) and African (34% Met allele) populations separately and also in comparison with Mexican (63% Met allele) population. In conclusion, the distribution of the Val108/158Met polymorphism distinguishes the Mexican population studied from other populations, but it is necessary to increase the size of the sample to get more conclusive results.     

Presence and quantity of dehydroalanine in histidine ammonia-lyase from Pseudomonas putida

Dehydroalanine is present in the histidine ammonia-lyase (histidase) from Pseudomonas putida ATCC 12633 as shown by reaction of purified enzyme with K14CN or NaB3H4 and subsequent identification of [14C]aspartate or [3H]alanine, respectively, following acid hydrolysis of the labeled protein. When labeling with cyanide was conducted under denaturing conditions, 4 mol of [14C]cyanide was incorporated per mol of enzyme (Mr 220 000), equivalent to one dehydroalanine residue being modified per subunit in this protein composed of four essentially identical subunits. In native enzyme, inactivation of catalytic activity by cyanide was complete when 1 mol of [14C]cyanide had reacted per mol of histidase, suggesting that modification of any one of the four dehydroalanine residues in the tetrameric enzyme was sufficient to prevent catalysis at all sites. Loss of activity on treatment with cyanide could be blocked by the addition of the competitive inhibitor cysteine or substrate if Mn2+ was also present. Cross-linking of native enzyme with dimethyl suberimidate produced no species larger than tetramer, thereby eliminating the possibility that an aggregation phenomenon might explain why only one-fourth of the dehydroalanyl residues was modified by cyanide during inactivation. A labeled tryptic peptide was isolated from enzyme inactivated with [14C]cyanide. Its composition was different from that of a tryptic peptide previously isolated from other histidases and shown to contain a highly reactive and catalytically important cysteine residue. Such a finding indicates the dehydroalanine group is distinct from the active site cysteine. Treatment of crude extracts with [14C]cyanide and purification of the inactive enzyme yielded labeled protein that release [14C]aspartate on acid hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemical modification of cysteine residues is a misleading indicator of their status as active site residues in the vitamin K-dependent gamma-glutamyl carboxylation reaction

The enzymatic activity of the vitamin K-dependent proteins requires the post-translational conversion of specific glutamic acids to gamma-carboxy-glutamic acid by the integral membrane enzyme, gamma-glutamyl carboxylase. Whether or not cysteine residues are important for carboxylase activity has been the subject of a number of studies. In the present study we used carboxylase with point mutations at cysteines, chemical modification, and mass spectrometry to examine this question. Mutation of any of the free cysteine residues to alanine or serine had little effect on carboxylase activity, although C343A mutant carboxylase had only 38% activity compared with that of wild type. In contrast, treatment with either thiol-reactive reagent 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, disodium salt, or sodium tetrathionate, caused complete loss of activity. We identified the residues modified, using matrix-assisted laser desorption/ionization time of flight mass spectrometry, as Cys(323) and Cys(343). According to our results, these residues are on the cytoplasmic side of the microsomal membrane, whereas catalytic residues are expected to be on the lumenal side of the membrane. Carboxylase was partially protected from chemical modification by factor IXs propeptide. Although all mutant carboxylases bound propeptide with normal affinity, chemical modification caused a >100-fold decrease in carboxylase affinity for the consensus propeptide. We conclude that cysteine residues are not directly involved in carboxylase catalysis, but chemical modification of Cys(323) and Cys(343) may disrupt the three-dimensional structure, resulting in inactivation.     

Identification and characterization of a putative active site for peptide methionine sulfoxide reductase (MsrA) and its substrate stereospecificity

Peptide methionine sulfoxide reductases (MsrA) from many different organisms share a consensus amino acid sequence (GCFWG) that could play an important role in their active site. Site-directed single substitution of each of these amino acids except glycines in the yeast MsrA resulted in total loss of enzyme activity. Nevertheless, all the recombinant MsrA mutants and native proteins had a very similar circular dichroism spectrum. The demonstration that either treatment with iodoacetamide or replacement of the motif cysteine with serine leads to inactivation of the enzyme underscores the singular importance of cysteine residues in the activity of MsrA. The recombinant yeast MsrA was used for general characterization of the enzyme. Its K(m) value was similar to the bovine MsrA and appreciably lower than the K(m) of the bacterial enzyme. Also, it was shown that the enzymatic activity increased dramatically with increasing ionic strength. The recombinant yeast MsrA activity and the reduction activity of free methionine sulfoxide(s) were stereoselective toward the L-methionine S-sulfoxide and S-methyl p-tolyl sulfoxide. It was established that a methionine auxotroph yeast strain could grow on either form of L-methionine sulfoxide.     

Identification of two cysteine residues involved in the binding of UDP-GalNAc to UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 1 (GalNAc-T1).

Biosynthesis of mucin-type O-glycans is initiated by a family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases, which contain several conserved cysteine residues among the isozymes. We found that a cysteine-specific reagent, p-chloromercuriphenylsulfonic acid (PCMPS), irreversibly inhibited one of the isozymes (GalNAc-T1). Presence of either UDP-GalNAc or UDP during PCMPS treatment protected GalNAc-T1 from inactivation, to the same extent. This suggests that GalNAc-T1 contains free cysteine residues interacting with the UDP moiety of the sugar donor. For the functional analysis of the cysteine residues, several conserved cysteine residues in GalNAc-T1 were mutated individually to alanine. All of the mutations except one resulted in complete inactivation or a drastic decrease in the activity, of the enzyme. We identified only Cys212 and Cys214, among the conserved cysteine residues in GalNAc-T1, as free cysteine residues, by cysteine-specific labeling of GalNAc-T1. To investigate the role of these two cysteine residues, we generated cysteine to serine mutants (C212S and C214S). The serine mutants were more active than the corresponding alanine mutants (C212A and C214A). Kinetic analysis demonstrated that the affinity of the serine-mutants for UDP-GalNAc was decreased, as compared to the wild type enzyme. The affinity for the acceptor apomucin, on the other hand, was essentially unaffected. The functional importance of the introduced serine residues was further demonstrated by the inhibition of all serine mutant enzymes with diisopropyl fluorophosphate. In addition, the serine mutants were more resistant to modification by PCMPS. Our results indicate that Cys212 and Cys214 are sites of PCMPS modification, and that these cysteine residues are involved in the interaction with the UDP moiety of UDP-GalNAc.     

Catechol-O-methyltransferase: effects of the val108met polymorphism on protein turnover in human cells

A single nucleotide polymorphism in the human COMT (catechol-O-methyltransferase) gene has been associated with increased risk for breast cancer and several CNS diseases and disorders. The G to A polymorphism causes a valine (val) to methionine (met) substitution at codon 108 soluble - (S)/158 membrane - (MB)-COMT, generating alleles encoding high and low-activity forms of the enzyme, COMT H and COMT L, respectively. Tissues and cells with a COMT LL genotype have decreased COMT activity compared to COMT HH cells. Previously, we reported that the decreased activity was due to decreased amounts of S-COMT L protein in human hepatocytes. In this study, we investigated the role of S-COMT protein synthesis and turnover as determinates of reduced COMT protein in COMT LL compared to COMT HH cells. No association between S-COMT protein synthesis and COMT genotype was detected. Using a pulse-chase protocol, the half-life of S-COMT H was determined to be 4.7 days, which was considerably longer than expected from the half-lives of other phase 2 enzyme proteins. The half-life of S-COMT L compared to S-COMT H protein was significantly shorter at 3.0 days, but the difference was affected by the medium used during the chase period. These results suggest that increased turnover may contribute to reduced COMT activity in cells and tissues from COMT LL individuals. Subtle differences appear to be able to affect the stability of the S-COMT L protein, and this may contribute to the differences observed in epidemiological studies on the association of this polymorphism with breast cancer risk.     

Mutant acyl-coenzyme A:cholesterol acyltransferase 1 devoid of cysteine residues remains catalytically active.

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) plays important roles in cellular cholesterol homeostasis and in the early stages of atherosclerosis. ACAT1 is an integral membrane protein with multiple transmembrane domains. Human ACAT1 contains nine cysteine residues; its activity is severely inhibited by various thiol-specific modification reagents including p-chloromercuribenzene sulfonic acid, suggesting that certain cysteine residue(s) might be near or at the active site. We constructed various ACAT1 mutants that contained either single cysteine to alanine substitution at various positions, contained a reduced number of cysteines, or contained no cysteine at all. Each of these mutants retained 20% or more of the wild-type ACAT activity. Therefore, cysteine is not essential for ACAT catalysis. For the cysteine-free enzyme, its basic kinetic properties and intracellular localization in Chinese hamster ovary cells were shown to be very similar to those of the wild-type enzyme. The availability of the cysteine-free ACAT1 will facilitate future ACAT structure function studies. Additional studies show that Cys467 is one of the major target sites that leads to p-chloromercuribenzene sulfonic acid-mediated ACAT1 inactivation, suggesting that Cys467 may be near the ACAT active site(s).     

Subcellular distribution of a factor inactivating tyrosine aminotransferase. Study of its mechanism and relationship to different forms of the enzyme.

The subcellular distribution of a tyrosine aminotransferase inactivating factor in rat liver has been investigated. Most of its activity is associated with plasma membranes, with minor amounts in mitochondria and endoplasmatic reticulum. The factor is also found in kidney and inactivates the enzyme reversibly in presence of cysteine, most likely by modification of -SH groups. ATP counteracts this inactivation only, when crude enzyme extracts are inactivated by purified subcellular fractions or when the purified enzyme is inactivated in presence of liver or kidney cortex homogenates. The relationship of this inactivation to reported different forms of the enzyme has been investigated. Form I of three different forms, that can be obtained by hydroxyl-apatite chromatography, is readily inactivated, form III can be partly converted to form I by incubation in presence of purified plasma membranes. The relationship of these findings to a possible multistep mechanism in the turnover of the enzyme discussed.     

Identification in Saccharomyces cerevisiae of a new stable variant of alkyl hydroperoxide reductase 1 (Ahp1) induced by oxidative stress.

Yeasts lacking cytoplasmic superoxide dismutase (Cu,Zn-SOD) activity are permanently subjected to oxidative stress. We used two-dimensional PAGE to examine the proteome pattern of Saccharomyces cerevisiae strains lacking Cu,Zn-SOD. We found a new stable form of alkyl hydroperoxide reductase 1 (Ahp1) with a lower isoelectric point. This form was also present in wild type strains after treatment with tert-butyl hydroperoxide. In vitro enzyme assays showed that Ahp1p had lower specific activity in strains lacking Cu,Zn-SOD. We studied three mutants presenting a reduced production of the low pI variant under oxidative stress conditions. Two of the mutants (C62S and S59D) were totally inactive, thus suggesting that the acidic form of Ahp1p may only appear when the enzyme is functional. The other mutant (S59A) was active in vitro and was more resistant to inactivation by tert-butyl hydroperoxide than the wild type enzyme. Furthermore, the inactivation of Ahp1p in vitro is correlated with its conversion to the low pI form. These results suggest that in vivo during some particular oxidative stress (alkyl hydroperoxide treatment or lack of Cu,Zn-SOD activity but not hydrogen peroxide treatment), the catalytic cysteine of Ahp1p is more oxidized than cysteine-sulfenic acid (a natural occurring intermediate of the enzymatic reaction) and that cysteine-sulfinic acid or cysteine-sulfonic acid variant may be inactive.     

Differences in thiol groups and multiple forms of rat liver alcohol dehydrogenase

Rat liver alcohol dehydrogenase was analyzed for multiple forms by gel-electrophoresis and for protein thiol groups by differential car?ymethylation before and after reduction. Differences in protein SH-groups were associated with different electrophoretic forms of the enzyme. In the form with highest mobility all cysteine residues were free to alkylation but in forms with lower mobilities some residues were unreactive. Three cysteine residues likely to be affected are reported. Different combinations of a few alternative SS-bridges, superficially located and of no importance in catalytic activity, are suggested to constitute one explanation to the multiplicity of the protein.