The oxidation of yeast alcohol dehydrogenase-1 by hydrogen peroxide in vitro

Yeast alcohol dehydrogenase (YADH) plays an important role in the conversion of alcohols to aldehydes or ketones. YADH-1 is a zinc-containing protein, and it accounts for the major part of ADH activity in growing baker's yeast. To gain insight into how oxidative modification of the enzyme affects its function, we exposed YADH-1 to hydrogen peroxide in vitro and assessed the oxidized protein by LC-MS/MS analysis of proteolytic cleavage products of the protein and by measurements of enzymatic activity, zinc release, and thiol/thiolate loss. The results illustrated that Cys43 and Cys153, which reside at the active site of the protein, could be selectively oxidized to cysteine sulfinic acid (Cys-SO2H) and cysteine sulfonic acid (Cys-SO3H). In addition, H2O2 induced the formation of three disulfide bonds: Cys43-Cys153 in the catalytic domain, Cys103-Cys111 in the noncatalytic zinc center, and Cys276-Cys277. Therefore, our results support the notion that the oxidation of cysteine residues in the zinc-binding domain of proteins can go beyond the formation of disulfide bond(s); the formation of Cys-SO2H and Cys-SO3H is also possible. Furthermore, most methionines could be oxidized to methionine sulfoxides. Quantitative measurement results revealed that, among all the cysteine residues, Cys43 was the most susceptible to H2O2 oxidation, and the major oxidation products of this cysteine were Cys-SO2H and Cys-SO3H. The oxidation of Cys43 might be responsible for the inactivation of the enzyme upon H2O2 treatment.     

Identification of a peroxide-sensitive redox switch at the CXXC motif in the human mitochondrial branched chain aminotransferase

The human mitochondrial branched chain aminotransferase isoenzyme (hBCATm) must be stored in a reducing environment to remain active. Oxidation or labeling of hBCATm with sulfhydryl reagents results in enzyme inhibition. In this study, we investigated both the structural and biochemical basis for the sensitivity of hBCATm to these reagents. In its native form, hBCATm has two reactive cysteine residues which were identified as Cys315 and Cys318 using iodinated beta-(4-hydroxyphenyl)ethyl maleimide. These are located in the large domain of the homodimer, about 10 A from the active site. The crystal structures show evidence for a thiol-thiolate hydrogen bond between Cys315 and Cys318. Under oxidizing conditions, these cysteine residues can reasonably form a disulfide bond because of the short distance between the sulfur atoms (3.09-3.46 A), requiring only a decrease of 1.1-1.5 A. In addition to Cys315 playing a structural role by anchoring Tyr173, which in the ketimine form increases access to the active site, our evidence indicates that these cysteine residues act as a redox switch in hBCATm. Electrospray ionization mass spectrometry analysis and UV-Vis spectroscopic studies of 5,5'-dithiobis(2-nitrobenzoic acid) labeled hBCATm showed that during labeling, an intrasubunit disulfide bond was formed in a significant portion of the protein. Furthermore, it was established that reaction of hBCATm with H2O2 abolished its activity and resulted in the formation of an intrasubunit disulfide bond between Cys315 and Cys318. Addition of dithiothreitol completely reversed the oxidation and restored activity. Therefore, the results demonstrate that there is redox-linked regulation of hBCATm activity by a peroxide sensitive CXXC center. Future studies will determine if this center has an in vivo role in the regulation of branched chain amino acid metabolism.     

An intramolecular disulfide bridge as a catalytic switch for serotonin N-acetyltransferase

Serotonin N-acetyltransferase (EC. 2.3.1.87) (AA-NAT) is a melatonin rhythm-generating enzyme in pineal glands. To establish a melatonin rhythm, AA-NAT activity is precisely regulated through several signaling pathways. Here we show novel regulation of AA-NAT activity, in which an intramolecular disulfide bond may function as a switch for the catalysis. Recombinant AA-NAT activity was irreversibly inhibited by N-ethylmaleimide (NEM) in an acetyl-CoA-protected manner. Oxidized glutathione or dissolved oxygen reversibly inhibited AA-NAT in an acetyl-CoA-protected manner. To identify the cysteine residues responsible for the inhibition, AA-NAT was first oxidized with dissolved oxygen, treated with NEM, reduced with dithiothreitol, and then labeled with [(14)C]NEM. Cys(61) and Cys(177) were specifically labeled in an acetyl-CoA-protected manner. The AA-NAT with the Cys(61) to Ala and Cys(177) to Ala double substitutions (C61A/C177A-AA-NAT) was fully active but did not exhibit sensitivity to either oxidation or NEM, whereas the AA-NATs with only the single substitutions retained about 40% of these sensitivities. An intramolecular disulfide bond between Cys(61) and Cys(177) formed upon oxidation and cleaved upon reduction was identified. Furthermore, C61A/C177A-AA-NAT expressed in COS7 cells was relatively insensitive to H(2)O(2)-evoked oxidative stress, whereas wild-type AA-NAT was strongly inhibited under the same conditions. These results indicate that the formation and cleavage of the disulfide bond between Cys(61) and Cys(177) produce the active and inactive states of AA-NAT. It is possible that intracellular redox conditions regulate AA-NAT activity through switching via an intramolecular disulfide bridge.     

Identification and reactivity of the catalytic site of pig liver thioltransferase

The active site cysteine of pig liver thioltransferase was identified as Cys22. The kinetics of the reaction between Cys22 of the reduced enzyme and iodoacetic acid as a function of pH revealed that the active site sulfhydryl group had a pKa of 2.5. Incubation of reduced enzyme with [1-14C]cysteine prevented the inactivation of the enzyme by iodoacetic acid at pH 6.5, and no stable protein-cysteine disulfide was found when the enzyme was separated from excess [1-14C]cysteine, suggesting an intramolecular disulfide formation. The results suggested a reaction mechanism for thioltransferase. The thiolated Cys22 first initiates a nucleophilic attack on a disulfide substrate, resulting in the formation of an unstable mixed disulfide between Cys22 and the substrate. Subsequently, the sulfhydryl group at Cys25 is deprotonated as a result of micro-environmental changes within the active site domain, releasing the mixed disulfide and forming an intramolecular disulfide bond. Reduced glutathione, the second substrate, reduces the intramolecular disulfide forming a transient mixed disulfide which is then further reduced by glutathione to regenerate the reduced enzyme and form oxidized glutathione. The rate-limiting step for a typical reaction between a disulfide and reduced glutathione is proposed to be the reduction of the intramolecular disulfide form of the enzyme by reduced glutathione.     

Thioredoxin-dependent enzymatic activation of mercaptopyruvate sulfurtransferase. An intersubunit disulfide bond serves as a redox switch for activation

Rat 3-mercaptopyruvate sulfurtransferase (MST) contains three exposed cysteines as follows: a catalytic site cysteine, Cys(247), in the active site and Cys(154) and Cys(263) on the surface of MST. The corresponding cysteine to Cys(263) is conserved in mammalian MSTs, and Cys(154) is a unique cysteine. MST has monomer-dimer equilibrium with the assistance of oxidants and reductants. The monomer to dimer ratio is maintained at approximately 92:8 in 0.2 m potassium phosphate buffer containing no reductants under air-saturated conditions; the dimer might be symmetrical via an intersubunit disulfide bond between Cys(154) and Cys(154) and between Cys(263) and Cys(263), or asymmetrical via an intersubunit disulfide bond between Cys(154) and Cys(263). Escherichia coli reduced thioredoxin (Trx) cleaved the intersubunit disulfide bond to activate MST to 2.3- and 4.9-fold the levels of activation of dithiothreitol (DTT)-treated and DTT-untreated MST, respectively. Rat Trx also activated MST. On the other hand, reduced glutathione did not affect MST activity. E. coli C35S Trx, in which Cys(35) was replaced with Ser, formed some adducts with MST and activated MST after treatment with DTT. Thus, Cys(32) of E. coli Trx reacted with the redox-active cysteines, Cys(154) and Cys(263), by forming an intersubunit disulfide bond and a sulfenyl Cys(247). A consecutively formed disulfide bond between Trx and MST must be cleaved for the activation. E. coli C32S Trx, however, did not activate MST. Reduced Trx turns on a redox switch for the enzymatic activation of MST, which contributes to the maintenance of cellular redox homeostasis.     

Formation of a covalent complex between methylguanine methyltransferase and DNA via disulfide bond formation between the active site cysteine and a thiol-containing analog of guanine.

DNA repair methyltransferases (MTases) remove methyl or other alkyl groups from the O6 position of guanine or the O4 position of thymine by transfering the group to an active site cysteine. In order to trap an MTase-DNA complex via a disulfide bond, 2'-deoxy-6-(cystamine)-2-aminopurine (d6Cys2AP) was synthesized and incorporated into oligonucleotides. d6Cys2AP has a disulfide bond within an alkyl chain linked to the 6 position of 2,6-diaminopurine, which disulfide can be reduced to form a free thiol. Addition of human MTase to reduced oligonucleotide resulted in a protein-DNA complex that was insensitive to denaturation by SDS and high salt, but which readily dissociated in the presence of dithiothreitol. Formation of this complex was prevented by methylation of the active site cysteine. Evidence that the active site cysteine is directly involved in disulfide bond formation was obtained by N-terminal sequencing of peptides that remained associated with DNA after proteolysis of the complex.     

Studies on the function of yeast protein disulfide isomerase in renaturation of proteins.

Renaturation of two enzymes lacking disulfide bonds, citrate synthase (CS), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and another protein containing disulfide bonds, lysozyme (LZM), were studied in order to dissect the possible chaperone function from the isomerase function of yeast protein disulfide isomerase (PDI). Our findings suggest no independent chaperone activity of yeast PDI with respect to the two enzymes lacking disulfide bonds, GAPDH and CS, since neither of these enzymes required PDI for renaturation. In contrast, a high level of renaturation of LZM was observed in the presence of PDI. Renaturation of LZM involved formation and rearrangement of disulfide bonds. Additional studies using LZM as a substrate were done to examine the role of cysteine residues in the two active sites of PDI. Studies with a series of cysteine to serine mutants and truncation mutants of yeast PDI revealed that the two active sites of PDI were not equal in activity. An intramolecular disulfide bond in at least one active site of PDI was required for the oxidation of reduced LZM. The first cysteine in each active site was necessary for disulfide bond rearrangement, i.e., isomerization, in LZM, while the second cysteine was not.     

Respiratory chain strongly oxidizes the CXXC motif of DsbB in the Escherichia coli disulfide bond formation pathway

Escherichia coli DsbB has four essential cysteine residues, among which Cys41 and Cys44 form a CXXC redox active site motif and the Cys104-Cys130 disulfide bond oxidizes the active site cysteines of DsbA, the disulfide bond formation factor in the periplasm. Functional respiratory chain is required for the cell to keep DsbA oxidized. In this study, we characterized the roles of essential cysteines of DsbB in the coupling with the respiratory chain. Cys104 was found to form the inactive complex with DsbA under respiration-defective conditions. While DsbB, under normal aerobic conditions, is in the oxidized state, having two intramolecular disulfide bonds, oxidation of Cys104 and Cys130 requires the presence of Cys41-Cys44. Remarkably, the Cys41-Cys44 disulfide bond is refractory to reduction by a high concentration of dithiothreitol, unless the membrane is solubilized with a detergent. This reductant resistance requires both the respiratory function and oxygen, since Cys41-Cys44 became sensitive to the reducing agent when membrane was prepared from quinone- or heme-depleted cells or when a membrane sample was deaerated. Thus, the Cys41-Val-Leu-Cys44 motif of DsbB is kept both strongly oxidized and strongly oxidizing when DsbB is integrated into the membrane with the normal set of respiratory components.     

Essential cysteine residues for human RNase κ catalytic activity

Human RNase κ is an endoribonuclease expressed in almost all tissues and organs and belongs to a highly conserved protein family bearing representatives in all metazoans. To gain insight into the role of cysteine residues in the enzyme activity or structure, a recombinant active form of human RNase κ expressed in Pichia pastoris was treated with alkylating agents and dithiothreitol (DTT). Our results showed that the human enzyme is inactivated by DDT, while it remains fully active in the presence of alkylating agents. The unreduced recombinant protein migrates on SDS/PAGE faster than the reduced form. This observation in combination with the above findings indicated that human RNase κ does not form homodimers through disulfide bridges, and cysteine residues are not implicated in RNA catalysis but participate in the formation of intramolecular disulfide bond(s) essential for its ribonucleolytic activity. The role of the cysteine residues was further investigated by expression and study of Cys variants. Ribonucleolytic activity experiments and SDS/PAGE analysis of the wild-type and mutant proteins under reducing and non-reducing conditions demonstrated that Cys7, Cys14 and Cys85 are not essential for RNase activity. On the other hand, replacement of Cys6 or Cys69 with serine led to a complete loss of catalytic activity, indicating the necessity of these residues for maintaining an active conformation of human RNase κ by forming a disulfide bond. Due to the absolute conservation of these cysteine residues, the Cys6-Cys69 disulfide bond is likely to exist in all RNase κ family members.     

Initial steps in assembly of microfibrils. Formation of disulfide-cross-linked multimers containing fibrillin-1

Fibrillins are the major constituents of extracellular microfibrils. How fibrillin molecules assemble into microfibrils is not known. Sequential extractions and pulse-chase labeling of organ cultures of embryonic chick aortae revealed rapid formation of disulfide-cross-linked aggregates containing fibrillin-1. These results demonstrated that intermolecular disulfide bond formation is an initial step in the assembly process. To identify free cysteine residues available for intermolecular cross-linking, small recombinant peptides of fibrillin-1 harboring candidate cysteine residues were analyzed. Results revealed that the first four cysteine residues in the unique N terminus form intramolecular disulfide bonds. One cysteine residue (Cys(204)) in the first hybrid domain of fibrillin-1 was found to occur as a free thiol and is therefore a good candidate for intermolecular disulfide bonding in initial steps of the assembly process. Furthermore, evidence indicated that the comparable cysteine residue in fibrillin-2 (Cys(233)) also occurs as a free thiol. These free cysteine residues in fibrillins are readily available for intermolecular disulfide bond formation, as determined by reaction with Ellman's reagent. In addition to these major results, the cleavage site of the fibrillin-1 signal peptide and the N-terminal sequence of monomeric authentic fibrillin-1 from conditioned fibroblast medium were determined.     

Functional characterization of ERp18, a new endoplasmic reticulum-located thioredoxin superfamily member

Native disulfide bond formation in the endoplasmic reticulum is a critical process in the maturation of many secreted and outer membrane proteins. Although a large number of proteins have been implicated in this process, it is clear that our current understanding is far from complete. Here we describe the functional characterization of a new 18-kDa protein (ERp18) related to protein-disulfide isomerase. We show that ERp18 is located in the endoplasmic reticulum and that it contains a single catalytic domain with an unusual CGAC active site motif and a probable insertion between beta3 and alpha3 of the thioredoxin fold. From circular dichroism and NMR measurements, ERp18 is well structured and undergoes only a minor conformational change upon dithioldisulfide exchange in the active site. Guanidinium chloride denaturation curves indicate that the reduced form of the protein is more stable than the oxidized form, suggesting that it is involved in disulfide bond formation. Furthermore, in vitro ERp18 possesses significant peptide thiol-disulfide oxidase activity, which is dependent on the presence of both active site cysteine residues. This activity differs from that of the human PDI family in that under standard assay conditions it is limited by substrate oxidation and not by enzyme reoxidation. A putative physiological role for Erp18 in native disulfide bond formation is discussed.     

Structural determinants of substrate access to the disulfide oxidase Erv2p

Erv2p is a small, dimeric FAD-dependent sulfhydryl oxidase that generates disulfide bonds in the lumen of the endoplasmic reticulum. Mutagenic and structural studies suggest that Erv2p uses an internal thiol-transfer relay between the FAD-proximal active site cysteine pair (Cys121-Cys124) and a second cysteine pair (Cys176-Cys178) located in a flexible, substrate-accessible C-terminal tail of the adjacent dimer subunit. Here, we demonstrate that Cys176 and Cys178 are the only amino acids in the tail region required for disulfide transfer and that their relative positioning within the tail peptide is important for activity. However, intragenic suppressor mutations could be isolated that bypass the requirement for Cys176 and Cys178. These mutants were found to disrupt Erv2p dimerization and to increase the activity of Erv2p for thiol substrates such as glutathione. We propose that the two Erv2p subunits act together to direct the disulfide transfer to specific substrates. One subunit provides the catalytic domain composed of the active site cysteine residues and the FAD cofactor, while the second subunit appears to have two functions: it facilitates disulfide transfer to substrates via the tail cysteine residues, while simultaneously shielding the active site cysteine residues from non-specific reactions.     

Importance of cysteine residues for the stability and catalytic activity of human pancreatic beta cell glucokinase

The low-affinity glucose phosphorylating enzyme glucokinase has the function of a physiological glucose sensor in pancreatic beta cells and in liver. In contrast to the high-affinity hexokinase types I-III glucokinase shows extraordinary sensitivity toward SH group oxidizing compounds. To characterize the function of sulfhydryl groups cysteine residues in the vicinity of the sugar binding site (Cys 213, Cys 220, Cys 230, Cys 233, and Cys 252) as well as cysteine residues a distance from the active site (Cys 364, Cys 371, and Cys 382), they were replaced in human beta cell glucokinase by serine through site-directed mutagenesis. Controlled proteolysis of wild-type glucokinase by proteinase K revealed that the SH group oxidizing agent alloxan can induce the formation of multiple intramolecular disulfide bridges corresponding to a double-band pattern of glucokinase protein in nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The formation of intramolecular disulfide bridges altered the mobility of the protein. None of the cysteine mutations could prevent the formation of the 49-kDa glucokinase conformation after alloxan treatment. The cysteine mutants Cys 233, Cys 252, and Cys 382 showed nearly complete loss of catalytic activity, whereas the V(max) values of the Cys 213, Cys 220, Cys 364, and Cys 371 mutants were decreased by 30-60%. Only the Cys 230 mutant showed kinetic characteristics comparable to those of wild-type glucokinase. The sensitivity of the Cys 213, Cys 230, Cys 364, and Cys 371 mutants toward alloxan-induced inhibition of enzyme activity was up to 10-fold lower compared with wild-type glucokinase. d-Glucose and dithiotreitol provided protection against alloxan-induced inhibition of wild-type glucokinase and all catalytically active cysteine mutants. Conclusively our data demonstrate the functional significance of the cysteine residues of beta cell glucokinase for both structural instability of the enzyme and catalytic function. Knowledge of sensitive cysteine targets may help to develop strategies that improve glucokinase enzyme function under conditions of oxidative stress.     

Redox properties of the tissue factor Cys186-Cys209 disulfide bond

TF (tissue factor) is a transmembrane cofactor that initiates blood coagulation in mammals by binding Factor VIIa to activate Factors X and IX. The cofactor can reside in a cryptic configuration on primary cells and de-encryption may involve a redox change in the C-terminal domain Cys(186)-Cys(209) disulfide bond. The redox potential of the bond, the spacing of the reduced cysteine thiols and their oxidation by TF activators was investigated to test the involvement of the dithiol/disulfide in TF activation. A standard redox potential of -278 mV was determined for the Cys(186)-Cys(209) disulfide of recombinant soluble TF. Notably, ablating the N-terminal domain Cys(49)-Cys(57) disulfide markedly increased the redox potential of the Cys(186)-Cys(209) bond, suggesting that the N-terminal bond may be involved in the regulation of redox activity at the C-terminal bond. Using As(III) and dibromobimane as molecular rulers for closely spaced sulfur atoms, the reduced Cys(186) and Cys(209) sulfurs were found to be within 3-6 ? (1 ?=0.1 nm) of each other, which is close enough to reform the disulfide bond. HgCl2 is a very efficient activator of cellular TF and activating concentrations of HgCl2-mediated oxidation of the reduced Cys(186) and Cys(209) thiols of soluble TF. Moreover, PAO (phenylarsonous acid), which cross-links two cysteine thiols that are in close proximity, and MMTS (methyl methanethiolsulfonate), at concentrations where it oxidizes closely spaced cysteine residues to a cystine residue, were efficient activators of cellular TF. These findings further support a role for Cys(186) and Cys(209) in TF activation.     

Evidence for activation of tissue factor by an allosteric disulfide bond

Tissue Factor (TF) is the mammalian plasma membrane cofactor responsible for initiation of blood coagulation. Binding of blood coagulation factor VIIa to TF activates the serine proteinase zymogens factors IX and X by limited proteolysis leading to the formation of a thrombin and fibrin meshwork that stabilizes the thrombus. TF on the plasma membrane of cells resides mostly in a cryptic configuration, which rapidly transforms into an active configuration in response to certain stimuli. The extracellular part of TF consists of two fibronectin type III domains. The disulfide bond in the membrane proximal domain (Cys186-Cys209) is atypical for domains of this type in that it links adjacent strands in the same beta sheet, what we have called an allosteric bond. Ablation of the allosteric disulfide by mutating both cysteine residues severely impairs procoagulant activity. The thiol-alkylating agents N-ethylmaleimide and methyl methanethiolsulfonate block TF activation by ionomycin, while the thiol-oxidizing agent HgCl2 and dithiol cross-linkers promote activation. TF activation could not be explained by exposure of phosphatidylserine on the outer leaflet of the plasma membrane. Cryptic TF contained unpaired cysteine thiols that were depleted upon activation, and de-encryption was associated with a change in the conformation of the membrane-proximal domain. These findings imply that the Cys186-Cys209 disulfide bond is reduced in the cryptic form of TF and that activation involves formation of the disulfide. It is likely that formation of this disulfide bond changes the conformation of the domain that facilitates productive binding of factors IX and X.     

The roles of surface loop insertions and disulfide bond in the stabilization of thermophilic WF146 protease

Thermophilic WF146 protease possesses four surface loop insertions and a disulfide bond, resembling its psychrophilic (subtilisins S41 and S39) and mesophilic (subtilisins SSII and sphericase) homologs. Deletion of the insertion 3 (positions 193-197) or insertion 4 (positions 210-221) of WF146 protease resulted in a significant decrease of the enzyme stability. In addition, substitution of the residues Pro211 and Ala212 or residue Glu221 which localized in the vicinity of a Ca(2+) binding site of the enzyme by the corresponding residues in subtilisin S41 remarkably reduced the half-life of the enzyme at 70 degrees C, suggesting that the three residues contributed to the thermostability of the enzyme, probably by enhancing the affinity of enzyme to Ca(2+). In the presence of dithiothreitol, the WF146 protease suffered excessive autolysis, indicating that the Cys52-Cys65 disulfide bond played a critical role in stabilizing the WF146 protease against autolysis. The autolytic cleavage sites of the WF146 protease were identified to locate between residues Asn63-Gly64 and Cys65-Ala66 by N-terminal amino acid analysis of the autolytic product. It was noticed that the effect of the autolytic cleavage at Asn63-Gly64 could be compensated by the disulfide bond Cys52-Cys65 under non-reducing condition, and the disulfide bond cross-linked autolytic product remained active. The apparent stabilization effect of the disulfide bond Cys52-Cys65 in the WF146 protease might provide a rational basis for improving the stability of subtilase against autolysis by protein engineering.     

An intra-dimeric crosslink of large subunits of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase is formed by oxidation of cysteine 247

Crystals of the hexadecameric form of ribulose-bisphosphate carboxylase used to solve the structure of the enzyme are composed of protein substantially crosslinked by a disulfide bond between pairs of large subunits. Conditions leading to the selective formation of dimers of the large subunits are described. The stability and specificity of the intra-dimeric crosslink was used to confirm that only one cysteine residue, Cys247 of neighboring large subunits, is involved in the bridge. The ability to generate this disulfide selectively, or alternatively replace the cysteine by site-directed mutagenesis, has led us to conclude that there is no effect of these changes on any of the critical kinetic parameters of the enzyme. The benign effect of the oxidation indicates that the crystal structures of the ribulose-bisphosphate carboxylase, particularly of the active site, are a true representation of the native enzyme.     

A novel member of the protein disulfide oxidoreductase family from Aeropyrum pernix K1: structure, function and electrostatics

The formation of disulfide bonds between cysteine residues is a rate-limiting step in protein folding. To control this oxidative process, different organisms have developed different systems. In bacteria, disulfide bond formation is assisted by the Dsb protein family; in eukarya, disulfide bond formation and rearrangement are catalyzed by PDI. In thermophilic organisms, a potential key role in disulfide bond formation has recently been ascribed to a new cytosolic Protein Disulphide Oxidoreductase family whose members have a molecular mass of about 26 kDa and are characterized by two thioredoxin folds comprising a CXXC active site motif each. Here we report on the functional and structural characterization of ApPDO, a new member of this family, which was isolated from the archaeon Aeropyrum pernix K1. Functional studies have revealed that ApPDO can catalyze the reduction, oxidation and isomerization of disulfide bridges. Structural studies have shown that this protein has two CXXC active sites with fairly similar geometrical parameters typical of a stable conformation. Finally, a theoretical calculation of the cysteine pK(a) values has suggested that the two active sites have similar functional properties and each of them can impart activity to the enzyme. Our results are evidence of functional similarity between the members of the Protein Disulphide Oxidoreductase family and the eukaryotic enzyme PDI. However, as the different three-dimensional features of these two biological systems strongly suggest significantly different mechanisms of action, further experimental studies will be needed to make clear how different three-dimensional structures can result in systems with similar functional behavior.     

Cysteine reactivity and thiol-disulfide interchange pathways in AhpF and AhpC of the bacterial alkyl hydroperoxide reductase system

AhpC and AhpF from Salmonella typhimurium undergo a series of electron transfers to catalyze the pyridine nucleotide-dependent reduction of hydroperoxide substrates. AhpC, the peroxide-reducing (peroxiredoxin) component of this alkyl hydroperoxidase system, is an important scavenger of endogenous hydrogen peroxide in bacteria and acts through a reactive, peroxidatic cysteine, Cys46, and a second cysteine, Cys165, that forms an active site disulfide bond. AhpF, a separate disulfide reductase protein, regenerates AhpC every catalytic cycle via electrons from NADH which are transferred to AhpC through a tightly bound flavin and two disulfide centers, Cys345-Cys348 and Cys129-Cys132, through putative large domain movements. In order to assess cysteine reactivity and interdomain interactions in both proteins, a comprehensive set of single and double cysteine mutants (replacing cysteine with serine) of both proteins were prepared. Based on 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) and AhpC reactivity with multiple mutants of AhpF, the thiolate of Cys129 in the N-terminal domain of AhpF initiates attack on Cys165 of the intersubunit disulfide bond within AhpC for electron transfer between proteins. Cys348 of AhpF has also been identified as the nucleophile attacking the Cys129 sulfur of the N-terminal disulfide bond to initiate electron transfer between these two redox centers. These findings support the modular architecture of AhpF and its need for domain rotations for function, and emphasize the importance of Cys165 in the reductive reactivation of AhpC. In addition, two new constructs have been generated, an AhpF-AhpC complex and a "twisted" form of AhpF, in which redox centers are locked together by stable disulfide bonds which mimic catalytic intermediates.     

Sulfur shuffle: modulating enzymatic activity by thiol-disulfide interchange

The facile modulation of biological processes is an important goal of biological chemists. Here, a general strategy is presented for controlling the catalytic activity of an enzyme. This strategy is demonstrated with ribonuclease A (RNase A), which catalyzes the cleavage of RNA. The side-chain amino group of Lys41 donates a hydrogen bond to a nonbridging oxygen in the transition state for RNA cleavage. Replacing Lys41 with a cysteine residue is known to decrease the value of k(cat)/K(m) by 10(5)-fold. Forming a mixed disulfide between the side chain of Cys41 of K41C RNase A and cysteamine replaces the amino group and increases k(cat)/K(m) by 10(3)-fold. This enzyme, which contains a mixed disulfide, is readily deactivated by dithiothreitol. Forming a mixed disulfide between the side chain of Cys41 and mercaptopropyl phosphate, which is designed to place a phosphoryl group in the active site, decreases activity by an additional 25-fold. This enzyme, which also contains a mixed disulfide, is reactivated in the presence of dithiothreitol and inorganic phosphate (which displaces the pendant phosphoryl group from the active site). An analogous control mechanism could be installed into the active site of virtually any enzyme by replacing an essential residue with a cysteine and elaborating the side chain of that cysteine into appropriate mixed disulfides.