Reactivities of neutral and cationic forms of 2,2''-dipyridyl disulphide towards thiolate anions. Detection of differences between the active centres of actinidin, papain and ficin by a three-protonic-state reactivity probe.

The second-order rate constants (k) for the reactions of 2,2'-dipyridyl disulphide (pKa2,45) with 2-mercaptoethanol (pKa9.6) and with benzimidazol-2-ylmethanethiol (pKa values 5.6 and 8.3) were determined at 25 degrees C at I 0.1 by stopped-flow spectral analysis over a wide range of pH. These were used to calculate the pH-independent second-order rate constants (k) for the reactions of neutral 2,2'-dipyridyl disulphide and of its monocation with the 2-mercaptoethanol thiolate anion (associated pKa9.6) and with the benzimidazol-2-ylmethanethiol zwitterion (associated pKa5.6). For both thiolate ions, the rate-enhancement factor (kmonocation/kneutral disulphide) is about 1.5x10(3). The dependence on pH in acidic media of k for the reaction of 2,2'-dipyridyl disulphide with actinidin, the thiol proteinase from Actinidia chinensis, was shown to differ from the forms of pH-dependence observed for the analogous reactions with papain (EC 3.4.22.2) and ficin (3.4.22.3). The reactivity of the 2,2'-dipyridyl disulphide dication and its apparent sensitivity to the presence and location of a positive charge in the attacking thiol are discussed.     

Evidence that the active centre of chymopapain A is different from the active centres of some other cysteine proteinases and that the Br?nsted coefficient (beta nuc.) for the reactions of thiolate anions with 2,2'-dipyridyl disulphide may be decreased by reagent protonation.

The active centres of chymopapains A and B (jointly designated EC 3.4.22.6) and papaya (Carica papaya L.) peptidase A were investigated by using 2,2'-dipyridyl disulphide and 5,5'-dithiobis-(2-nitrobenzoic acid) as thiol-specific reactivity probes. Whereas the first active-centre pKa values for chymopapain B and papaya peptidase A are less than 5, is as the case for papain (EC 3.4.22.2) and ficin (EC 3.4.22.3), that for chymopapain A is about 6.8. The reason why the reactions of thiols of pKa approx. 6.5 with 2.2'-dipyridyl disulphide are essentially pH-independent in the pH range around the thiol pKa is delineated. The value of the Brønsted coefficient (beta nuc.) for the reactions of thiolate ions with the 2,2'-dipyridyl disulphide monocation appears to be smaller than its value for the corresponding reactions with the neutral disulphide.     

A kinetic method for the study of solvent environments of thiol groups in proteins involving the use of a pair of isomeric reactivity probes and a differential solvent effect. Investigation of the active centre of ficin by using 2,2''- and 4,4''- dipyridyl disulphides as reactivity probes.

1. Whereas the second-order rate constants for the reaction of the thiolate ion of 2-mercaptoethanol with 4,4'-dipyridyl disulphide (k4PDS) and with 5,5'-dithiobis-2-nitrobenzoate dianion increase with decreasing dielectric constant of the solvent, or remain unchanged, the rate constant for the analogous reaction with 2,2'-dipyridyl disulphide (k2PDS) decreases. This anomalous solvent effect and other unusual physicochemical properties of 2,2'-dipyridyl disulphide are discussed. 2. The differential effect of solvent on the reactions of thiolate ion with the 2,2'- and 4,4'-dipyridyl disulphides is shown to provide a method of characterizing solvent environments of thiol groups in proteins by a reactivity-probe method that should not suffer from the usual drawback associated with the existence of steric or binding effects of unknown magnitude. Application of the method to ficin (EC 3.4.22.3) suggests that its active-centre thiol group resides in a relatively hydrophobic environment. 3. The pH-k profile for the reaction of ficin with 4,4'-dipyridyl disulphide is reported.     

Structural studies on the reaction of isopenicillin N synthase with the truncated substrate analogues delta-(L-alpha-aminoadipoyl)-L-cysteinyl-glycine and delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-alanine

Isopenicillin N synthase (IPNS), a non-heme iron(II)-dependent oxidase, catalyzes conversion of the tripeptide delta-(l-alpha-aminoadipoyl)-l-cysteinyl-d-valine (ACV) to bicyclic isopenicillin N (IPN), concomitant with the reduction of dioxygen to two molecules of water. Incubation of the "truncated"substrate analogues delta-(l-alpha-aminoadipoyl)-l-cysteinyl-glycine (ACG) and delta-(l-alpha-aminoadipoyl)-l-cysteinyl-d-alanine (ACA) with IPNS has previously been shown to afford acyclic products, in which the substrate cysteinyl residue has undergone a two-electron oxidation. We report X-ray crystal structures for the anaerobic IPNS/Fe(II)/ACG and IPNS/Fe(II)/ACA complexes, both in the absence and presence of the dioxygen analogue nitric oxide. The overall protein structures are very similar to those of the corresponding IPNS/Fe(II)/ACV complexes; however, significant differences are apparent in the vicinity of the active site iron. The structure of the IPNS/Fe(II)/ACG complex reveals that the C-terminal carboxylate of this substrate is oriented toward the active site iron atom, apparently hydrogen-bonded to an additional water ligand at the metal; this is a different binding mode to that observed in the IPNS/Fe(II)/ACV complex. ACA binds to the metal in a manner that is intermediate between those observed for ACV and ACG. The addition of NO to these complexes initiates conformational changes such that both the IPNS/Fe(II)/ACG/NO and IPNS/Fe(II)/ACA/NO structures closely resemble the IPNS/Fe(II)/ACV/NO complex. These results further demonstrate the feasibility of metal-centered rearrangements in catalysis by non-heme iron enzymes and provide insight into the delicate balance between hydrophilic-hydrophobic interactions and steric effects in the IPNS active site.     

High level expression in Escherichia coli of a fungal gene under the control of strong promoters

A recent report (Patino et al., (1989) FEMS Microbiol. Lett. 58, 139-144) described the low level expression, in Escherichia coli, of the Isopenicillin N Synthase (IPNS) gene from Cephalosporium acremonium under the control of strong promoters. We report here our work on the expression of the IPNS gene. Plasmids containing the IPNS gene under the control of the trp or trc promoters directed synthesis of high levels of active IPNS in E. coli. Constitutive and inductive high level IPNS expression systems have been developed. Importantly, the expression vectors do not encode beta-lactamase so IPNS activity can be determined directly by biological assays. Analysis by nmr verified that the IPNS produced from these expression systems catalysed the conversion of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (LLD-ACV) to isopenicillin N in high yield.     

Sulfite oxidation by iron-grown cells of Thiobacillus ferrooxidans at pH 3 possibly involves free radicals, iron, and cytochrome oxidase

Thiobacillus ferrooxidans cells grown on ferrous iron oxidized sulfite to sulfate at pH 3, possibly by a free radical mechanism involving iron and cytochrome oxidase. A purely chemical system with low concentrations of Fe3+ simulated the T. ferrooxidans system. Metal chelators, ethylenediamine tetraacetic acid (EDTA), 4,5-dihydroxy-1-3-benzene disulfonic acid (Tiron), o-phenanthroline, and 2,2'-dipyridyl, inhibited both sulfite oxidation systems, but the T. ferrooxidans system was inhibited only after the initial brief oxygen consumption. EDTA and Tiron, strong chelators of Fe3+, inhibited the oxidation at lower concentrations than o-phenanthroline and 2,2'-dipyridyl, strong chelators of Fe2+. Inhibition of Fe3+-catalyzed sulfite oxidation by EDTA and Tiron was instant, but the inhibition by o-phenanthroline and dipyridyl was briefly delayed, presumably for the reduction of Fe3+ to Fe2+. Mannitol, a free radical scavenger, inhibited both systems to the same extent. Cyanide and azide inhibited only the T. ferrooxidans system, suggesting a role of cytochrome oxidase. It is proposed that sulfite is oxidized by a free radical mechanism initiated by Fe3+ on the cell surface of T. ferrooxidans. Cytochrome oxidase is possibly involved in the regeneration of Fe3+ from Fe2+ by the normal Fe2+-oxidizing system of T. ferrooxidans.     

Quantitative analysis of Penicillium chrysogenum Wis54-1255 transformants overexpressing the penicillin biosynthetic genes

The low penicillin-producing, single gene copy strain Wis54-1255 was used to study the effect of overexpressing the penicillin biosynthetic genes in Penicillium chrysogenum. Transformants of Wis54-1255 were obtained with the amdS expression-cassette using the four combinations: pcbAB, pcbC, pcbC-penDE, and pcbAB-pcbC-penDE of the three penicillin biosynthetic genes. Transformants showing an increased penicillin production were investigated during steady-state continuous cultivations with glucose as the growth-limiting substrate. The transformants were characterized with respect to specific penicillin productivity, the activity of the two pathway enzymes delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) and isopenicillin N synthetase (IPNS) and the intracellular concentration of the metabolites: delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV), bis-delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (bisACV), isopenicillin N (IPN), glutathione (GSH), and glutathione disulphide (GSSG). Transformants with the whole gene cluster amplified showed the largest increase in specific penicillin productivity (r(p))-124% and 176%, respectively, whereas transformation with the pcbC-penDE gene fragment resulted in a decrease in r(p) of 9% relative to Wis54-1255. A marked increase in r(p) is clearly correlated with a balanced amplification of both the ACVS and IPNS activity or a large amplification of either enzyme activity. The increased capacity of a single enzyme occurs surprisingly only in the transformants where all the three biosynthetic genes are overexpressed but is not found within the group of pcbAB or pcbC transformants. The indication of the pcbAB and pcbC genes being closely regulated in fungi might explain why high-yielding strains of P. chrysogenum have been found to contain amplifications of a large region including the whole penicillin gene cluster and not single gene amplifications. Measurements of the total ACV concentration showed a large span of variability, which reflected the individual status of enzyme overexpression and activity found in each strain. The ratio ACV:bisACV remained constant, also at high ACV concentrations, indicating no limitation in the capacity of the thioredoxin-thioredoxin reductase (TR) system, which is assumed to keep the pathway intermediate LLD-ACV in its reduced state. The total GSH pool was at a constant level of approx. 5.7 mM in all cultivations.     

Physicochemical characterization of the microbial Fe2+ chelator proferrorosamine from Pseudomonas roseus fluorescens.

The microbial chelating compound proferrorosamine A, produced by Pseudomonas roseus fluorescens, formed a complex with Fe2+ of which the apparent stability constant was found to be 10(23). The following order of increasing stability constants of metal complexes with proferrorosamine was established as: Ba2+, Ca2+, Mg2+, Mn2+ less than Hg2+ less than Zn2+ less than Pb2+ less than Co2+ less than Cu2+ congruent to Fe2+ less than Ni2+. Only Ni(2+)-proferrorosamine had a stability constant which was established as: Ba2+, Ca2+, Mg2+, Mn2+ less than Hg2+ less than Zn2+ less than Pb2+ less than Co2+ less than Cu2+ congruent to Fe2+ less than Ni2+. Only Ni(2+)-proferrorosamine had a stability constant which was ca 32 times higher than Fe(2+)-proferrorosamine. Because of the production of proferrorosamine the growth of Ps. roseus fluorescens was not inhibited in iron limiting media by the addition of 0.15 mmol/l of the weaker chemical Fe2+ chelator 2,2'-dipyridyl. This contrasted with the proferrorosamine-negative mutant K2 and Ps. stutzeri, which only produces Fe(3+)-chelating siderophores. Furthermore, it was found that proferrorosamine was able to dissolve Fe2+ from stainless steel. These results show that proferrorosamine is a strong and selective Fe2+ chelator which could be used as an alternative for the toxic 2,2'-dipyridyl to control lactic acid fermentations.     

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.     

Redox modification of sodium-calcium exchange activity in cardiac sarcolemmal vesicles

Na-Ca exchange activity in bovine cardiac sarcolemmal vesicles was stimulated up to 10-fold by preincubating the vesicles with 1 microM FeSO4 plus 1 mM dithiothreitol (DTT) in a NaCl medium. The increase in activity was not reversed upon removing the Fe and DTT. Stimulation of exchange activity under these conditions was completely blocked by 0.1 mM EDTA or o-phenanthroline; this suggests that the production of reduced oxygen species (H2O2, O2-.,.OH) during Fecatalyzed DTT oxidation might be involved in stimulating exchange activity. In agreement with this hypothesis, the increase in exchange activity in the presence of Fe-DTT was inhibited 80% by anaerobiosis and 60% by catalase. H2O2 (0.1 mM) potentiated the stimulation of Na-Ca exchange by Fe-DTT under both aerobic and anaerobic conditions; H2O2 also produced an increase in activity in the presence of either FeSO4 (1 microM) or DTT (1 mM), but it had no effect on activity by itself. Superoxide dismutase did not block the effects of Fe-DTT on exchange activity; however, the generation of O2-. by xanthine oxidase in the presence of an oxidizable substrate stimulated activity more than 2-fold. Hydroxyl radical scavenging agents (mannitol, sodium formate, sodium benzoate) did not attenuate the stimulation of activity observed with Fe-H2O2. Exchange activity was also stimulated by the simultaneous presence of glutathione (GSH; 1-2 mM) and glutathione disulfide (GSSG; 1-2 mM). Neither GSH nor GSSG was effective by itself and either 0.1 mM EDTA or o-phenanthroline blocked the effects on transport activity of the combination of GSH + GSSG. Treatment of the GSH and GSSG solutions with Chelex ion-exchange resin to remove contaminating transition metal ions reduced (by 40%) the degree of stimulation observed with GSH + GSSG. Full stimulating activity was restored to the Chelex-treated GSH and GSSG solutions by the addition of 1 microM Fe2+; Cu2+ was less effective than Fe2+ whereas Co2+ and Mn2+ were without effect. In the presence of 1 microM Fe2+, GSH alone produced a slight increase in transport activity, but this was markedly enhanced by the addition of Chelex-treated GSSG. The results indicate that stimulation of exchange activity requires the presence of both a reducing agent (DTT, GSH, O-.2, or Fe2+) and an oxidizing agent (H2O2, GSSG, and perhaps O2) and that the effects of these agents are mediated by metal ions (e.g. Fe2+).(ABSTRACT TRUNCATED AT 400 WORDS)     

Intermediate Mr cytosolic components potentiate hepatic 5''-deiodinase activation by thiols.

The role in the activation of microsomal 5'-deiodinase (5'-DI) of rat hepatic cytosolic components of Mr approx. 13,000 (Fraction B) was studied in the presence of various concentrations of thiol compounds such as dithiothreitol (DTT), dihydrolipoamide (DHLA), GSH, and 2-mercaptoethanol (2-ME). Although Fraction B (which was prepared by gel filtration to exclude GSH and GSSG) had no intrinsic 5'-DI activity, could not stimulate microsomal 5'-DI activity in the absence of added thiol and did not contain GSH as a mixed disulphide, it could produce a 3-fold increase in the maximal deiodinase activity achievable with DTT as well as other thiols, with the order being the same as the activation potency of these thiols in the absence of Fraction B (i.e. DHLA greater than DTT greater than 2-ME greater than GSH). These observations suggest that: a component of cytosolic Fraction B, designated 'deiodination factor B' (DFB), operates as an efficient intermediary to enhance activation of microsomal 5'-DI by thiols through a mechanism independent of GSH; thiols may participate in a non-specific thiol-disulphide exchange with inactive (oxidized) DFB to convert it into an active form that contains one or more thiol groups and is more effective than GSH or other thiols in facilitating the re-activation of inactive (oxidized) microsomal 5'-DI thiol (ESI) to its active state (ESH).     

Dithiothreitol induces the sacrificial antioxidant property of human serum albumin in a metal-catalyzed oxidation and gamma-irradiation system

The species *OH or H2O2 are produced by both metal-catalyzed oxidation (MCO) of reducing equivalents and gamma-irradiation. Intact or Cys-34-modified human serum albumin (HSA) was significantly degraded in the MCO system containing dithiothreitol (DTT) as electron donor, but as long as it lasted, HSA prohibited *OH or H2O2 from initiating molecular damage of DNA. However, in the GSH and ascorbate (nonthiol) MCO system, HSA was not sacrificially degraded, and indeed accelerated the formation of DNA strand breaks. In the y-irradiation system producing *OH from H2O, only DTT attenuated the generation of DNA strand breaks by HSA. It did not degrade more H2O2 in the presence of reduced GSH (thiol-linked peroxidase) than in its absence. Therefore it would seem that in an MCO system, the antioxidant activity of HSA depends on the effectiveness of reducing equivalents to induce exposure of a functional group scavenging the *OH or H2O2 species, by reduction of its disulfide-bonds. In the presence of DTT, disulfide bonds in HSA were quantitatively reduced to cysteinyl residues but not significantly reduced by ascorbate or GSH. In conclusion, the antioxidant activity of HSA in the D     

Distinctive iron requirement of tryptophan 5-monooxygenase: TPH1 requires dissociable ferrous iron

A peripheral type of tryptophan 5-monooxygenase (EC 1.14.16.4), TPH1, is very unstable in vitro, but the inactivation was reversible and full reactivation occurs upon anaerobic incubation with a high concentration of dithiothreitol (DTT, 15 mM). In this study, distinctive iron requirement of TPH1 was revealed through analysis of the enzyme's inactivation and activation by DTT. For this purpose, all the glasswares, plastics, Sephadex G-25 gels, and reagents including protein solutions had been treated with metal chelators, and apo-TPH was prepared by treatment with EDTA. Apo-TPH thus prepared exclusively required free Fe2+ for its catalytic activity; 10(-8) M was enough under the strict absence of Fe3+ but 10(-12) M was too low. No other metal ions including Fe3+ were effective. It appeared that Fe3+ bound to the enzyme with a higher affinity than Fe2+, resulting in the inactivation. Ascorbate, a non-thiol reducing agent, did not substitute DTT in the activation of TPH1, but enhanced the Fe2+-dependent activity of apo-TPH as effectively as DTT. Thus, the DTT-activation was essentially substituted by preparation of apo-TPH by the EDTA treatment and the assay of apo-TPH in the presence of Fe2+ and ascorbate. The activation of TPH1 by incubation with DTT was accompanied by exposure of 9 sulfhydryls out of the total 10 cysteine residues, but the cleavage of disulfide bonds seemed not to be crucial, even if it occurred. The effect of DTT was substituted by some other sulfhydryls whose structure was analogous to that of commonly used metal chelators. Based on these observations, the following dual roles of DTT are proposed: (1) in the activation of TPH, DTT removes inappropriate bound iron (Fe3+) as a chelator, keeping Fe3+ away from the enzyme's binding site which needs to bind Fe2+ for the catalytic activity, and (2) in both the activation and reaction processes, DTT prevents oxidation of Fe2+ to Fe3+ as a reducing agent.     

Preparation of cathepsins B and H by covalent chromatography and characterization of their catalytic sites by reaction with a thiol-specific two-protonic-state reactivity probe. Kinetic study of cathepsins B and H extending into alkaline media and a rapid spectroscopic titration of cathepsin H at pH 3-4.

A procedure for the isolation of cathepsin B (EC 3.4.22.1) and of cathepsin H from bovine spleen involving covalent chromatography by thiol-disulphide interchange and ion-exchange chromatography was devised. The stabilities of both cathepsins in alkaline media are markedly temperature-dependent, and reliable kinetic data can be obtained at pH values up to 8 by working at 25 degrees C with a continuous spectrophotometric assay. Both enzyme preparations contain only one type of thiol group as judged by reactivity characteristics towards 2,2'-dipyridyl disulphide at pH values up to 8; in each case this thiol group is essential for catalytic activity. Cathepsin H was characterized by kinetic analysis of the reactions of its thiol group with 2,2'-dipyridyl disulphide in the pH range approx. 2-8 and the analogous study on cathepsin B [Willenbrock & Brocklehurst (1984) Biochem. J. 222, 805-814] was extended to include reaction at pH values up to approx. 8. Cathepsin H, like the other cysteine proteinases, was shown to contain an interactive catalytic-site system in which the nucleophilic character of the sulphur atom is maintained in acidic media. The considerable differences in catalytic site characteristics detected by this two-protonic-state reactivity probe between cathepsin B, cathepsin H, papain (EC 3.4.22.2) and actinidin (EC 3.4.22.14) are discussed. Reaction with 2,2'-dipyridyl disulphide in acidic media, which is known to provide a rapid spectrophotometric active centre titration for many cysteine proteinases, is applicable to cathepsin H. This is useful because other active-centre titrations have proved unsuitable in view of the relatively low reactivity of the thiol group in cathepsin H.     

Pathway kinetics and metabolic control analysis of a high-yielding strain of Penicillium chrysogenum during fed batch cultivations

A kinetic model representing the pathway for the biosynthesis of penicillin by P. chrysogenum has been developed. The model is capable of describing the flux through the biosynthetic pathway, and model simulations correspond well with measurements of intermediates and end products. One feature of the present model structure is that it assumes the kinetics of the enzyme isopenicillin N synthetase (IPNS) to be first order with respect to the dissolved oxygen concentration in the range of 0.070 to 0.18 mM (25% to 70% saturation with air). Thus, it indicates the importance that molecular oxygen has on the rate of the reaction catalyzed by this enzyme, and consequently as an enhancer of the specific rate of penicillin production. Using the kinetic model, metabolic control analysis (MCA) of the pathway was performed. The determined flux control coefficients suggested that, during the production phase, the flux is controlled by IPNS as this enzyme becomes saturated with tripeptide delta-(L-alpha-amino-adipyl)-L-cysteinyl-D-valine (LLD-ACV). In the simulations, oxygen was shown to be a bottleneck alleviator by stimulating the rate of IPNS which prevents the accumulation of LLD-ACV. As a consequence of this stimulation, the rate-controlling step was moved to another place in the pathway. (c) 1996 John Wiley & Sons, Inc.     

Differences in the chemical and catalytic characteristics of two crystallographically ''identical'' enzyme catalytic sites. Characterization of actinidin and papain by a combination of pH-dependent substrate catalysis kinetics and reactivity probe studies targeted on the catalytic-site thiol group and its immediate microenvironment.

The characteristics of actinidin (EC 3.4.22.14) and papain (EC 3.4.22.2), two cysteine proteinases whose catalytic-site regions appear to superimpose to a degree that approaches atomic co-ordinate accuracy of both crystal structures, were evaluated by determining (a) the pH-dependence in acid media of the acylation process of the catalytic act (k+2/Ks) using N alpha-benzoyl-L-arginine p-nitroanilide (L-Bz-Arg-Nan) as substrate and (b) the sensitivity of the reactivity of the catalytic-site thiol group and its pH-dependence to structural change in small, thiol-specific, two-protonic-state reactivity probes (2,2'-dipyridyl disulphide and methyl 2-pyridyl disulphide) where enzyme-probe contacts should be restricted to areas close to the catalytic site. Distortion of the catalytic sites of the two enzymes at pH less than 4 was evaluated over time-scales appropriate for both stopped-flow reactivity probe kinetics (less than or equal to 1-2 s) and steady-state substrate catalysis kinetics (3-5 min) by using the 2,2'-dipyridyl disulphide monocation as a titrant for non-distorted catalytic sites. This permitted a lower pH limit to be defined for valid kinetic analysis of both types. The behaviour of the enzymes at pH less than 4 requires a kinetic model in which the apparently biomolecular reaction of enzyme with probe reagent is separated from the process leading to loss of conformational integrity by a potentially reversible step. The acylation of actinidin with L-Bz-Arg-Nan in acidic media occurs in two protonic states, one produced by raising the pH across pKa less than 4 which probably characterizes the formation of -S-/-ImH+ ion pair (pKa approx. 3) and the other, of higher reactivity, produced by raising the pH across pKa 5.5, which may characterize rearrangement of catalytic-site geometry. The pH-dependence of the acylation of papain by L-Bz-Arg-Nan is quite different and is not influenced by protonic dissociation with pKa values in the range 5-6. The earlier conclusion that the acylation of papain depends on two protonic dissociations each with pKa approx. 4 was confirmed. This argument is now more firmly based because titration with 2,2'-dipyridyl disulphide permits the loss of conformational integrity to be taken into account in the analysis of the kinetic data at very low pH. Methyl 2-pyridyl disulphide was synthesized by reaction of pyridine-2-thione with methyl methanethiolsulphonate and its pKa at I = 0.1 was determined by spectral analysis at 307 nm to be 2.8.(ABSTRACT TRUNCATED AT 400 WORDS)     

Crystallographic studies on the binding of selectively deuterated LLD- and LLL-substrate epimers by isopenicillin N synthase

Isopenicillin N synthase (IPNS) is a non-heme iron(II) oxidase which catalyses the biosynthesis of isopenicillin N (IPN) from the tripeptide δ-l-α-aminoadipoyl-l-cysteinyl-d-valine (lld-ACV). Herein we report crystallographic studies to investigate the binding of a truncated lll-substrate in the active site of IPNS. Two epimeric tripeptides have been prepared by solution phase peptide synthesis and crystallised with the enzyme. δ-l-α-Aminoadipoyl-l-cysteinyl-d-2-amino-3,3-dideuteriobutyrate (lld-ACd₂Ab) has the same configuration as the natural substrate lld-ACV at each of its three stereocentres; its epimer δ-l-α-aminoadipoyl-l-cysteinyl-l-2-amino-3,3-dideuteriobutyrate (lll-ACd₂Ab) has the opposite configuration at its third amino acid. lll-ACV has previously been shown to inhibit IPNS turnover of its substrate lld-ACV; the all-protiated tripeptide δ-l-α-aminoadipoyl-l-cysteinyl-d-2-aminobutyrate (lld-ACAb) is a substrate for IPNS, being turned over to a mixture of penam and cepham products. Comparisons between the crystal structures of the IPNS:Fe(II):lld-ACd₂Ab and IPNS:Fe(II):lll-ACd₂Ab complexes offer a possible rationale for the previously observed inhibitory effects of lll-ACV on IPNS activity.     

Evidence that binding to the S2-subsite of papain may be coupled with catalytically relevant structural change involving the cysteine-25–histidine-159 diad. Kinetics of the reaction of papain with a two-protonic-state reactivity probe containing a hydrophobic side chain

A method is proposed by which site-specific reactivity probes that exhibit different reactivities in two ionization states can be used to detect association-activation phenomena that involve repositioning of acid/base groups in enzyme active centres. The pH-dependences of the apparent second-order rate constants (k) for the reactions of the thiol group of papain (EC 3.4.22.2) with a series of two-protonic-state reactivity probes are compared. The short-chain probes, 2,2'-dipyridyl disulphide and n-propyl 2-pyridyl disulphide, react at pH6 in adsorptive complexes and/or transition states with geometries that do not permit hydrogen-bonding of the pyridyl nitrogen atom with the active-centre imidazolium ion, as evidenced by the rate minima at pH6 and the rate maxima at pH4 provided by reagent protonation. Only when the probe molecule, e.g. 4-(N-aminoethyl 2'-pyridyl disulphide)-7-nitrobenzo-2-oxa-1,3-diazole [compound(III)], contains a long hydrophobic side chain is the reaction characterized by maximal rates at about pH6, as in the acylation step of the catalytic act (at pH6, k(compound III)/k(2,2'-dipyridyl disulphide) approximately 100). It is proposed that this striking difference in profile shape may result from binding of the hydrophobic side chain of compound (III) possibly in the S(2)-subsite of papain, which promotes a change in catalytic-site geometry involving repositioning of the imidazolium ion of histidine-159 and hydrogen-bonding with the N atom of the leaving group, as has been postulated to occur in the acylation step of substate hydrolysis.     

2,2′-Dipyridyl diselenide is a better antioxidant than other disubstituted diaryl diselenides

The aim of this study was to investigate the in vitro antioxidant activity of 2,2'-dipyridyl diselenide (e) by comparing this effect with m-trifluoromethyl-diphenyl diselenide (a), p-fluor-diphenyl diselenide (b), p-chloro-diphenyl diselenide (c), and p-methoxyl-diphenyl diselenide (d) in rat liver homogenate. We also investigated if the mechanisms involved in the antioxidant property of 2,2'-dipyridyl diselenide are the same that of other diselenides. Thiobarbituric acid reactive substances (TBARS) and protein carbonyl (PC) levels were determined in rat liver homogenate, as indicators of antioxidant activity. Dehydroascorbate (DHA) reductase- and glutathione S-transferase (GST)-like activities, 2,2'-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical-scavenging activities and the protection against the oxidation of Fe(2+) were determined to better understand the antioxidant property of compounds. δ-Aminolevulinic dehydratase (δ-ALA-D) activity was also carried out in rat liver homogenates, as a toxicological parameter. Compound e showed the highest potency in reducing TBARS (order of IC(50) values: e < b ≤ a < d ≤ c) and PC (order of IC(50) values: e < c ≤ b ≤ a < d) levels and lower potency in inhibiting δ-ALA-D activity than other diselenides. Compound e at all concentrations tested had no enzyme-mimetic property, but had radical-scavenging activity (≥5 μM) and protected against the oxidation of Fe(2+) (50 μM); while compounds a-d showed GST and DHA-mimetic activities and protected against the oxidation of Fe(2+), but had not radical-scavenging activities. This study indicates that (i) 2,2'-dipyridyl diselenide (e) had better in vitro antioxidant effect than other diselenides and lower inhibitory effect on δ-ALA-D activity, (ii) the presence of pyridine ring is responsible for the best antioxidant effect of this compound, and (iii) 2,2'-dipyridyl diselenide acts by different mechanisms of other diselenides.