Purification and characterization of a novel proteinase, chymopapain S

Chymopapain (EC 3.4.22.6) possesses an essential and a non-essential thiol group, but enzyme preparations produced hitherto always contained less than two thiol groups. Starting from commercial chymopapain or from papaya latex, we have prepared pure enzyme having two thiol groups, which is demanded by mechanistic investigations. The purification was performed by covalent chromatography on activated thiol-Sepharose column, which specifically binds thiol-containing proteins. Elution was effcted with cysteine in a stepwise manner, first at pH 5 then at pH 8. The pure enzyme had a molecular mass of24 700 as estimated by sodium dodecyl sulfate polyacrylamide gel-electrophoresis. Titration of the pure enzyme with 2,2'-dipyridyl disulfide at pH 9 exhibited a biphasic curve. This indicated that under the conditions employed the nonessential thiol group is more reactive than the essential one, which is a characteristic feature of chymopapain B. On the other hand, the magnitude of the rate constant of the same reaction at pH 4, as well as its pH-dependence, was characteristic of chymopapain A. The enzyme with the hybrid kinetic properties was denoted as chymopapain S. We conclude from the above findings that various forms of chymopapain can be classified by their reaction with 2,2'-dipyridyl disulfide.     

Reactivity of the essential thiol of Klebsiella aerogenes urease. Effect of pH and ligands on thiol modification

The kinetics of Klebsiella aerogenes urease inactivation by disulfide and alkylating agents was examined and found to follow pseudo-first-order kinetics. Reactivity of the essential thiol is affected by the presence of substrate and competitive inhibitors, consistent with a cysteine located proximal to the active site. In contrast to the results observed with other reagents, the rate of activity loss in the presence of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) saturated at high reagent concentrations, indicating that DTNB must first bind to urease before inactivation can occur. The pH dependence for the rate of urease inactivation by both disulfide and alkylating agents was consistent with an interaction between the thiol and a second ionizing group. The resulting macroscopic pKa values for the 2 residues are less than 5 and 12. Spectrophotometric studies at pH 7.75 demonstrated that 2,2'-dithiodipyridine (DTDP) modified 8.5 +/- 0.2 mol of thiol/mol of enzyme or 4.2 mol of thiol/mol of catalytic unit. With the slow tight binding competitive inhibitor phenyl-phosphorodiamidate (PPD) bound to urease, 1.1 +/- 0.1 mol of thiol/mol of catalytic unit were protected from modification. PPD-bound DTDP-modified urease could be reactivated by dialysis, consistent with the presence of one thiol per active site. Analogous studies at pH 6.1, using the competitive inhibitor phosphate, confirmed the presence of one protected thiol per catalytic unit. Under denaturing conditions, 25.5 +/- 0.3 mol of thiol/mol of enzyme (Mr = 211, 800) were modified by DTDP.     

Protein-thiol substitution or protein dethiolation by thiol/disulfide exchange reactions: the albumin model

Dethiolation experiments of thiolated albumin with thionitrobenzoic acid and thiols (glutathione, cysteine, homocysteine) were carried out to understand the role of albumin in plasma distribution of thiols and disulfide species by thiol/disulfide (SH/SS) exchange reactions. During these experiments we observed that thiolated albumin underwent thiol substitution (Alb-SS-X+RSH<-->Alb-SS-R+XSH) or dethiolation (Alb-SS-X+XSH<-->Alb-SH+XSSX), depending on the different pK(a) values of thiols involved in protein-thiol mixed disulfides (Alb-SS-X). It appeared in these reactions that the compound with lower pK(a) in mixed disulfide was a good leaving group and that the pK(a) differences dictated the kind of reaction (substitution or dethiolation). Thionitrobenzoic acid, bound to albumin by mixed disulfide (Alb-TNB), underwent rapid substitution after thiol addition, forming the corresponding Alb-SS-X (peaks at 0.25-1 min). In turn, Alb-SS-X were dethiolated by the excess nonprotein SH groups because of the lower pK(a) value in mixed disulfide with respect to that of other thiols. Dethiolation of Alb-SS-X was accompanied by formation of XSSX and Alb-SH up to equilibrium levels at 35 min, which were different for each thiol. Structures by molecular simulation of thiolated albumin, carried out for understanding the role of sulfur exposure in mixed disulfides in dethiolation process, evidenced that the sulfur exposure is important for the rate but not for determining the kind of reaction (substitution or dethiolation). Our data underline the contribution of SH/SS exchanges to determine levels of various thiols as reduced and oxidized species in human plasma.     

Hydrolytically induced allosteric change in the heavy chain of intact myosin involving nonessential thiol groups.

The two globular head portions, each bearing an active site, contain an uncleaved heavy chain when isolated by chymotrypsin from intact myosin. By specific labeling with radioactive N-ethylmaleimide the essential thiol 1 and thiol 2 groups were found to reside in this heavy chain. In intact myosin nonessential thiol 3 groups become the most reactive during ATP hydrolysis above 15 degrees C. These thiol 3 groups are located in a portion of the myosin heavy chain which appears as a fragment with an apparent molecular weight of 11 000 during proteolysis. The facts that this fragment is produced in an almost 1: 1 molar ratio with the head heavy chain and that it bears unblocked N-terminal amino groups whereas the heavy chain does not and is not contained in the rod portion of the myosin molecule indicate that it may orginate from the heavy chains in the neck region where the heads are joined to the rod. Since this fragment is removed by ion-exchange chromatography, it is not part of the functioning head and hence not involved in the active site. As its nonessential thiol 3 groups are rendered the most reactive of all thiol groups in the enzyme-product complex M**ADP.Pi, the hydrolytic step induces an allosteric conformational change in the neck region of intact myosin.     

Chymopapain A. Purification and investigation by covalent chromatography and characterization by two-protonic-state reactivity-probe kinetics, steady-state kinetics and resonance Raman spectroscopy of some dithioacyl derivatives.

Chymopapain A was isolated from the dried latex of papaya (Carica papaya) by ion-exchange chromatography followed by covalent chromatography by thiol-disulphide interchange. The latter procedure was used to produce fully active enzyme containing one essential thiol group per molecule of protein, to establish that the chymopapain A molecule contains, in addition, one non-essential thiol group per molecule and to recalculate the literature value of epsilon 280 for the enzyme as 36 000 M-1 X cm -1. The Michaelis parameters for the hydrolysis of L-benzoylarginine p-nitroanilide and of benzyloxy-carbonyl-lysine nitrophenyl ester at 25 degrees C, and I 0.1 at several pH values catalysed by chymopapain A, papaya proteinase omega, papain (EC 3.4.22.2) and actinidin (EC 3.4.22.14) were determined. Towards these substrates chymopapain A has kcat./km values similar to those of actinidin and of papaya proteinase omega and significantly lower than those of papain or ficin. The environment of the catalytic site of chymopapain A is markedly different from those of other cysteine proteinases studied to date, as evidenced by the pH-dependence of the second-order rate constant (k) for the reaction of the catalytic-site thiol group with 2,2'-dipyridyl disulphide. The striking bell-shaped component that is a characteristic feature of the reactions of S-/ImH+ (thiolate/imidazolium) ion-pair components of many cysteine-proteinase catalytic sites with the 2,2'-dipyridyl disulphide univalent cation is not present in the pH-k profile for the chymopapain A reaction. The result is consistent with the presence of an additional positive charge in, or near, the catalytic site that repels the cationic form of the probe reagent. Resonance Raman spectra were collected at pH values 2.5, 6.0 and 8.0 for each of the following dithioacyl derivatives of chymopapain A: N-benzoylglycine-, N-(Beta-phenylpropionl)glycine- and N-methoxycarbonylphenylalanylglycine-. The main conclusion of the spectral study is that in each case the acyl group binds as a single population known as conformer B in which the glycinic N atom is in close contact with the thiol S atom of the catalytic-site cysteine residue, as is the case also for papain and other cysteine proteinases studied. Thus the abnormal catalytic-site environment of chymopapain A detected by the reactivity-probe studies, which may have consequences for the acylation step of the catalytic act, does not perturb the conformation of the bound acyl group at the acyl-enzyme-intermediate stage of catalysis.     

Isolation and characterization of the four major cysteine-proteinase components of the latex of carica papaya L. reactivity characteristics towards 2,2′-dipyridyl disulfide of the thiol groups of papain,chymopapains A and B,and papaya peptidase A

High-quality spray-dried latex of Carica papaya L was fractionated by using SP-Sephadex-C50. The four major cysteine-proteinase components—papain(E.C.3.4.22.2), chymopapains A and B(jointly designated currently as E.C.3.4.22.6), and papaya peptidase A—were isolated and characterized by protein chemical methods and by study of their thiol groups using2,2′-dipyridyl disulfide as a two-protonic-state titrant and reactivity probe. Papain and papaya peptidase A each contain one thiol group/molecule, which in each case is part of the catalytic site, as evidenced by high reactivity toward2,2′-dipyridyl disulfide in acidic media. Chymopapains A and B each contain two thiol groups/molecule, only one of which is essential for catalytic activity. The reactivities of the thiol groups of these enzymes toward2,2′-dipyridyl disulfide at pH4 and10 and activity loss analysis by Tsou Chen-Lu plots each provides a ready means of distinguishing among the four cysteine proteinases. The nonessential thiol groups of chymopapains A and B readily undergo irreversible oxidation. The reactivity characteristics of the essential thiol groups of the four enzymes suggest the presence of somewhat similar interactive cysteine-histidine catalytic center systems in papain, papaya peptidase A, and chymopapain B but a different type of catalytic center environment in chymopapain A.     

Evidence for a thiol reagent inhibiting choline acetyltransferase by reacting with the thiol group of coenzyme A forming a potent ingibitor.

Evidence is presented for the thiol reagent methyl methanethiolsulfonate inhibiting choline acetyltransferase (EC 2.3.1.6), not by reaction with an enzymic thiol group, but by reaction with the thiol group of CoA. The resulting CoA methyl disulfide is a potent inhibitor of this enzyme. Its action is reversed competitively by acetyl CoA.     

On the molecular mechanism of maize phosphoenolpyruvate carboxylase activation by thiol compounds

Incubation of purified phosphoenolpyruvate carboxylase from Zea mays L. leaves with dithiothreitol resulted in an almost 2-fold increase in the enzymic activity. The activated enzyme showed the same affinity for its substrates and the same sensitivity with respect to malate and oxalacetate inhibition. The activation induced by dithiothreitol was reversed by diamide, an oxidant of vicinal dithiols, suggesting that the redox state of disulfide bonds of the enzyme may be important in the expression of the maximal catalytic activity.

Titration of thiol groups before and after activation of maize phosphoenolpyruvate carboxylase by dithiothreitol shows an increase of the accessible groups from 8 to 12 suggesting that the reduction of two disulfide bonds accompanied the activation. The thiols exposed by the treatment with dithiothreitol were available to reagents in nondenatured enzyme and two of them were reoxidized to a disulfide bond by diamide. It is concluded that the mechanism of phosphoenolpyruvate carboxylase activation by dithiothreitol involves the net reduction of two disulfide bonds in the enzyme.

     

Aromatic thiol pKa effects on the folding rate of a disulfide containing protein

The production of proteins via recombinant DNA technology often requires the in vitro folding of inclusion bodies, which are protein aggregates. To create a more efficient redox buffer for the in vitro folding of disulfide containing proteins, aromatic thiols were investigated for their ability to increase the folding rate of scrambled RNase A. Scrambled RNase A is fully oxidized RNase A with a relatively random distribution of disulfide bonds. The importance of the thiol pK(a) value was investigated by the analysis of five para-substituted aromatic thiols with pK(a) values ranging from 5.2 to 6.6. Folding was measured at pH 6.0 where the pK(a) value of the thiols would be higher, lower, or equal to the solution pH. Thus, relative concentrations of thiol and thiolate would vary across the series. At pH 6.0, the aromatic thiols increased the folding rate of RNase A by a factor of 10-23 over that observed for glutathione, the standard additive. Under optimal conditions, the apparent rate constant increased as the thiol pK(a) value decreased. Optimal conditions occurred when the concentration of protonated thiol in solution was approximately 2 mM, although the total thiol concentration varied considerably. The importance of the concentration of protonated thiol in solution can be understood based on equilibrium effects. Kinetic studies suggest that the redox buffer participates as the nucleophile and/or the center thiol in the key rate determining thiol disulfide interchange reactions that occur during protein folding. Aromatic thiols proved to be kinetically faster and more versatile than classical aliphatic thiol redox buffers.     

Reactivity of the essential thiol group of lactate dehydrogenase and substrate binding

1. The preparation of a derivative of pig heart lactate dehydrogenase in which the essential thiol group has been converted into an S-sulpho group is described. The derivative has unchanged s(20,w) and is catalytically inactive. 2. The rate of reaction of the essential thiol group is controlled by a system with a pK>9. 3. The essential thiol group is protected by NADH against reaction with maleimide. 4. Lactate dehydrogenase in which the essential thiol group has been converted into an S-sulpho group or alkylated with maleimide still binds one molecule of NADH/subunit but with a three- to four-fold diminished affinity. 5. The inhibited enzymes also bind one molecule of NAD(+)-sulphite complex/subunit but with affinity decreased 10(3)-10(4)-fold. 6. The inhibited enzymes fail to bind C(2) and C(3) molecules to give the ternary complexes enzyme-NAD(+)-pyruvate, enzyme-NADH-oxamate and enzyme-NADH-oxalate. The 1:1:1 stoicheiometry of the last-mentioned complex with the native enzyme was established by gel filtration. 7. Structures that account for these results are discussed.     

Catalysis of thiol/disulfide exchange: single-turnover reduction of protein disulfide-isomerase by glutathione and catalysis of peptide disulfide reduction

Protein disulfide-isomerase, a protein localized to the lumen of the endoplasmic reticulum of eukaryotic cells, catalyzes the posttranslational formation and rearrangement of protein disulfide bonds. As isolated from bovine liver, the enzyme contains 0.8 free sulfhydryl group per mole of protein monomer and 3.1 disulfide bonds. Single-turnover experiments in which the disulfide bonds of the native enzyme are reduced by glutathione reveal three distinct reduction steps corresponding to the sequential reduction of the three disulfide bonds. The fastest disulfide to be reduced undergoes a change in the rate-determining step with increasing GSH concentration from a step which is second-order with respect to GSH concentration to a step which is first-order in GSH concentration. The disulfide which is reduced at an intermediate rate displays kinetics that are first-order in GSH concentration, and the slowest disulfide to be reduced exhibits kinetics which are second-order in GSH concentration. The enzyme catalyzes the steady-state reduction of a disulfide-containing hexapeptide (CYIQNC) by GSH. Initial velocity kinetic experiments are consistent with a sequential addition of the substrates to the enzyme. Saturation behavior is not observed at high levels of both substrates (Km for GSH much greater than 14 mM, Km for CYIQNC much greater than 1 mM). Only one of the three disulfides appears to be kinetically competent in the steady-state reduction of CYIQNC by GSH. The second-order thiol/disulfide exchange reactions catalyzed by the enzyme are 400-6000-fold faster than the corresponding uncatalyzed reactions.     

Thiol/disulfide exchange between rabbit muscle phosphofructokinase and glutathione. Kinetics and thermodynamics of enzyme oxidation

Reversible thiol/disulfide exchange equilibria between rabbit muscle phosphofructokinase and glutathione redox buffers results in a dependence of the activity of the enzyme on the thiol to disulfide ratio of the redox buffer (Gilbert, H. F. (1982) J. Biol. Chem. 257, 12086-12091). The transition between fully reduced (active) and fully oxidized (inactive) enzyme is half complete at a [GSH]/[GSSG] ratio of 6.5 +/- 1 at pH 8.0 and 5.6 +/- 0.9 at pH 7.2. In the presence of excess GSSG approximately 40-50% of the activity is lost in a rapid process (k = 110 M-1 min-1), while the remaining activity is lost more slowly (k = 1.9 M-1 min-1). Two equivalents of radiolabeled glutathione are incorporated covalently, one coincident with each phase of inactivation. The most rapidly oxidized sulfhydryl group is also the most rapidly reduced by GSH in the reverse reaction (k = 150 M-1 min-1). Reduction of a more slowly reacting protein-glutathione mixed disulfide is required to regenerate the original activity (k = 0.33 M-1 min-1). The thiol/disulfide oxidation equilibrium constant (Kox) for the most rapidly oxidized sulfhydryl group is estimated to be 0.7 while that for the more slowly oxidized group is 6.1. The sulfhydryl group which is more easily oxidized kinetically is the more thermodynamically resistant to oxidation. The magnitude of the equilibrium constants for these reversible oxidations would suggest that the oxidation state (and activity) of phosphofructokinase would not be significantly affected by typical metabolic changes in the glutathione oxidation state in vivo.     

Chemical modification of the RTEM-1 thiol beta-lactamase by thiol-selective reagents: evidence for activation of the primary nucleophile of the beta-lactamase active site by adjacent functional groups

The RTEM-1 thiol beta-lactamase (Sigal, I.S., Harwood, B.G., Arentzen, R., Proc. Natl. Acad. Sci. U.S.A. 79:7157-7160, 1982) is inactivated by thiol-selective reagents such as iodoacetamide, methyl methanethiosulfonate, and 4,4'-dipyridyldisulfide, which modify the active site thiol group. The pH-rate profiles of these inactivation reactions show that there are two nucleophilic forms of the enzyme, EH2 and EH, both of which, by analogy with the situation with cysteine proteinases, probably contain the active site nucleophile in the thiolate form. The pKa of the active site thiol is therefore shown by the data to be below 4.0. This low pKa is thought to reflect the presence of adjacent functionality which stabilizes the thiolate anion. The low nucleophilicity of the thiolate in both EH2 and EH, with respect to that of cysteine proteinases and model compounds, suggests that the thiolate of the thiol beta-lactamase is stabilized by two hydrogen-bond donors. One of these, of pKa greater than 9.0, is suggested to be the conserved and essential Lys-73 ammonium group, while the identity of the other group, of pKa around 6.7, is less clear, but may be the conserved Glu-166 carboxylic acid. beta-Lactamase activity is associated with the EH2 form, and thus the beta-lactamase active site is proposed to contain one basic or nucleophilic group (the thiolate in the thiol beta-lactamase) and two acidic (hydrogen-bond donor) groups (one of which is likely to be the above-mentioned lysine ammonium group).     

The dark (oxidized) form of the light-activatable NADP-malate dehydrogenase from pea chloroplasts is catalytically active in the presence of guanidine-HCl

The dark form of NADP-malate dehydrogenase from pea leaves which has been shown to contain one disulfide bridge per subunit does not exhibit any catalytic activity in the absence of thiol reducing agents. Upon reduction of these disulfide bridges the enzyme becomes catalytically active. In this presentation, however, it is shown that the oxidized dark form of NADP-malate dehydrogenase becames catalytically competent when assayed in the presence of 200–250 mM guanidine-HCl. This guanidine-dependent activity of the oxidized enzyme is characterized by higher apparent K_m values for the substratres as compared to the reduced enzyme, but is still specific for NADPH. Up to 25% of the V_maxOf the reduced enzyme was obtained for the oxidized guanidine-activated NADP-malate dehydrogenase. The results suggest that the reduction of the regulatory disulfide is not essential for catalytic activity.     

Protein thiol modifications visualized in vivo

Thiol-disulfide interconversions play a crucial role in the chemistry of biological systems. They participate in the major systems that control the cellular redox potential and prevent oxidative damage. In addition, thiol-disulfide exchange reactions serve as molecular switches in a growing number of redox-regulated proteins. We developed a differential thiol-trapping technique combined with two-dimensional gel analysis, which in combination with genetic studies, allowed us to obtain a snapshot of the in vivo thiol status of cellular proteins. We determined the redox potential of protein thiols in vivo, identified and dissected the in vivo substrate proteins of the major cellular thiol-disulfide oxidoreductases, and discovered proteins that undergo thiol modifications during oxidative stress. Under normal growth conditions most cytosolic proteins had reduced cysteines, confirming existing dogmas. Among the few partly oxidized cytosolic proteins that we detected were proteins that are known to form disulfide bond intermediates transiently during their catalytic cycle (e.g., dihydrolipoyl transacetylase and lipoamide dehydrogenase). Most proteins with highly oxidized thiols were periplasmic proteins and were found to be in vivo substrates of the disulfide-bond-forming protein DsbA. We discovered a substantial number of redox-sensitive cytoplasmic proteins, whose thiol groups were significantly oxidized in strains lacking thioredoxin A. These included detoxifying enzymes as well as many metabolic enzymes with active-site cysteines that were not known to be substrates for thioredoxin. H₂O₂-induced oxidative stress resulted in the specific oxidation of thiols of proteins involved in detoxification of H₂O₂ and of enzymes of cofactor and amino acid biosynthesis pathways such as thiolperoxidase, GTP-cyclohydrolase I, and the cobalamin-independent methionine synthase MetE. Remarkably, a number of these proteins were previously or are now shown to be redox regulated.     

Reactive thiol groups in rat liver acid phosphatase

Dimeric rat liver acid phosphatase P1 of Mr 92,000 is inactivated by p-chloromercuribenzoate and fluorescein mercuriacetate (FMA). The enzyme is protected against the mercurials by the substrate analogue Pi. The reaction with FMA is accompanied by changes in absorbance at 495 nm and in fluorescence emission at 520 nm that are characteristic of reaction of this compound with thiol groups. Titration of P1 with FMA monitored by spectrophotometry or by fluorimetry indicated that equivalence is reached at an FMA/P1 ratio of 3. Since FMA can act as a bifunctional reagent, it is likely that P1 contains either 3 or 6 reactive thiol groups per molecule. Analysis of FMA inactivation/modification data by a statistical method suggests that of 6 reactive thiol groups, 2 are essential so that there are probably 3 thiol groups per subunit, one of which is located at the active site. If the total thiol number is 3, analysis suggests 1 essential thiol per subunit.     

Glucagon activation of the thiol:protein disulfide oxidoreductase in isolated, rat, hepatic microsomes

The hepatic, microsomal, thiol:protein disulfide oxidoreductase catalyzes the glutathione (GSH) reduction of protein disulfides to sulfhydryl groups. In the presence of physiological concentrations of glucagon this activity increased from 2.3 to 6.4 fold in isolated microsomes. The stimulation had a P50 for glucagon of 7.8 X 10(-10) M which was only observed at microsomal protein concentrations of less than 100 micrograms/ml and in the presence of a GSH reducing system. This latter observation suggests that the stimulation may be inhibited by the presence of oxidized glutathione. These data support the hypothesis that glucagon may act in part by stimulating the reduction of protein disulfides by the thiol:protein disulfide oxidoreductase.