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PubMed1979

The alkaline transition of swine pepsinogen.

At alkaline pH, swine pepsinogen is reversibly inactivated in a transition which involves the cooperative release of two protons from the molecule and is governed by a pK = 9. Stopped flow kinetic studies on the absorbance changes accompanying this reaction show that it can be resolved into two steps, with increasing pH; a slow conformational change, whose amplitude follows the ionisation curve of one group of pK = 9.9, followed by a rapid pH dependent conformational change, linked to a group of pK = 8.2. The pH dependence of the rate of the slow step is interpreted to show the presence of a protonated group which cannot ionise in the neutral form of the zymogen, but is in slow equilibrium with a form where it titrates with a pK 6.8. At the same time, a histidine in the amino terminal region of the protein becomes reactive to diethyl pyrocarbonate, suggesting this to be the group which triggers the reaction.

Animals↗
PubMed1986

Kinetic analysis of enzyme inactivation by an autodecaying reagent.

A simple method is described for the determination of both the pseudo-first-order rate constant and the second-order rate constant for enzyme inactivation by a chemical reagent which itself undergoes exponential decay. The validity of this method has been demonstrated in two test cases in which the labile diethyl pyrocarbonate was used to inactivate salicylate hydroxylase and bacterial luciferase.

Diethyl Pyrocarbonate↗
PubMed1981

S-2-hydroxyacylglutathione hydrolase (glyoxalase II): active-site mapping of a nonserine thiolesterase.

S-2-Hydroxyacylglutathione hydrolase (glyoxalase II) from rat erythrocytes is a specific thiolesterase. Chemical modification studies with phenylmethanesulfonic acid, N-ethylmaleimide, and 5,5'-dithiobis(2-nitrobenzoate) suggest that glyoxalase II does not have a serine or a cysteine residue at the active site. The effect of pH on the rate of hydrolysis of S-lactoylglutathione indicates the existence of an active-site residue, pK = 8.87, essential for binding of the substrate. Inactivation studies with phenylglyoxal implicate the existence of an active-site arginine residue that also is essential for binding of the substrate. The effects of pD on the rate of hydrolysis of S-lactoylglutathione in D2O give no evidence for general acid-general base catalysis. 1H NMR studies of the glyoxalase II catalyzed hydrolysis of S-mandeloylglutathione show no evidence for a carbanion (E1cB) mechanism. the catalytic role of glyoxalase II appears to involve direct nucleophilic attack of the thiol ester by an active-site histidine residue, based upon inactivation experiments using diethyl pyrocarbonate and photoinactivation with methylene blue.

Animals↗
PubMed1982

Protection of platelet surface bound factors IXa and VIII against specific inhibitors.

The rate of inactivation of factors IXa and VIII as well as of the complex formed by them on the surface of platelets, induced by specific inhibitors has been studied. After the formation of complex both factors became more protected against high molecular weight inhibitors. As evidenced by the apparent first order inactivation rate constants, after complex formation the velocity of IXa-antithrombin III + heparin reaction decreased to 1/20. Enhanced resistance toward the high molecular weight inhibitors is presumably due to a steric change, since the low molecular weight phenylmethyl sulfonyl fluoride and diethyl pyrocarbonate inhibited free and complexed factor IXa to the same extent.

Antibodies↗
PubMed1989

Redox properties of the diheme cytochrome c4 from Azotobacter vinelandii and characterisation of the two hemes by NMR, MCD and EPR spectroscopy.

From biphasic stopped-flow kinetic studies it has been established that the two heme centres of cytochrome c4 from Azotobacter vinelandii undergo redox change with [Co(terpy)2]3+/2+ (260 mV) at different rates. Rate constants for oxidation and reduction at pH 7.5 give reduction potentials for the two heme centres in agreement with previous values from spectrophotometric titrations (263 and 317 mV). From NMR studies on the fully reduced protein two sharp methyl methionine resonances are observed at -3.16 and -3.60 ppm, consistent with axial methionine coordination. On titration with [Fe(CN)6]3- the -3.16 ppm resonance is the first to disappear, and is assigned to the less positive reduction potential. Line-broadening effects are observed on partial oxidation, which are dominated by intermolecular processes in an intermediate time-range exchange process. The hemes of the oxidised protein are distinguishable by EPR g-values of 3.64 and 3.22. The former is of interest because it is at an unusually low field for histidine/methionine coordination, and has an asymmetric or ramp shape. The latter assigned to the low potential heme is similar to that of a cytochrome c551. The MCD spectra of the fully oxidised protein are typical of low-spin Fe(III) heme centres, with a negative peak at 710 nm characteristic of methionine coordination, and an NIR peak at 1900 nm characteristic of histidine/methionine (axial) coordination. Of the four histidines per molecule only two undergo diethyl pyrocarbonate (DEPC) modification.

Azotobacter↗
PubMed1994

Mutagenesis of rat liver arginase expressed in Escherichia coli: role of conserved histidines.

Rat liver arginase has been overexpressed in Escherichia coli using a T7-based expression system. The kinetic properties of the recombinant wild-type protein are essentially identical to those of the native rat liver enzyme. The recombinant wild-type protein contains six Mn(II) ions per trimer, in good agreement with results obtained with the fully active native enzyme. However, in contrast to the native enzyme which loses three Mn(II) per trimer upon extended dialysis, the recombinant protein binds Mn(II) tenaciously, and retains six Mn(II) per trimer even after extensive dialysis. Three histidine residues, corresponding to His101, His126, and His141 in the rat liver enzyme, are highly conserved in arginases from evolutionarily divergent species. The replacement of His101 and His126 with Asn by site-directed mutagenesis produced only modest effects on enzymatic activity when measured in the presence of Mn(II) ions. However, EDTA treatment of these mutant enzymes reduced activity to < 0.2% of that for the wild-type enzyme. The activity of wild-type enzyme and the His141 Asn mutant was unaffected by treatment with EDTA. Thus, His101 and His126 are proposed to be ligands to the binuclear Mn(II) center of the enzyme. The His141 Asn mutation produced an enzyme which, in contrast to the native, wild-type, His101 Asn, and His126 Asn arginases, was not inactivated by diethyl pyrocarbonate. These results suggest a catalytic role for His141.

Animals↗
PubMed1993

Molecular characterization of flavanone 3 beta-hydroxylases. Consensus sequence, comparison with related enzymes and the role of conserved histidine residues.

A heterologous cDNA probe from Petunia hybrida was used to isolate flavanone-3 beta-hydroxylase-encoding cDNA clones from carnation (Dianthus caryophyllus), china aster (Callistephus chinensis) and stock (Matthiola incana). The deduced protein sequences together with the known sequences of the enzyme from P. hybrida, barley (Hordeum vulgare) and snapdragon (Antirrhinum majus) enabled the determination of a consensus sequence which revealed an overall 84% similarity (53% identity) of flavanone 3 beta-hydroxylases from the different sources. Alignment with the sequences of other known enzymes of the same class and to related non-heme iron-(II) enzymes demonstrated the strict genetic conservation of 14 amino acids, in particular, of three histidines and an aspartic acid. The conservation of the histidine motifs provides strong support for the possible conservation of structurally similar iron-binding sites in these enzymes. The putative role of histidines as chelators of ferrous ions in the active site of flavanone 3 beta-hydroxylases was corroborated by diethyl-pyrocarbonate modification of the partially purified recombinant Petunia enzyme.

Amino Acid Sequence↗
PubMed1991

Isolation and characterization of a homogeneous isoenzyme of wheat germ acid phosphatase.

An acid phosphatase (orthophosphoric monoester phosphohydrolase, acid optimum; EC 3.1.3.2) isoenzyme from wheat germ was purified 7000-fold to homogeneity. The effect of wheat germ sources and their relationship to the isoenzyme content and purification behavior of acid phosphatases was investigated. Extensive information about the purification and stabilization of the enzyme is provided. The instability of isoenzymes in the latter stages of purification appeared to be the result of surface inactivation together with a sensitivity to dilution that could be partially offset by addition of Triton X-100 during chromatographic procedures. Added sulfhydryl protecting reagents had no effect on activity or stability, which was greatest in the pH range 4-7. The purified isoenzyme was homogeneous by polyacrylamide gel electrophoresis and exhibited the highest specific activity and turnover number reported for any acid phosphatase. The molecular weights of the pure isoenzyme and of related isoenzymes from wheat germ were found to be identical (58,000). The pure isoenzyme contained a single polypeptide chain and had a negligible carbohydrate content. The amino acid composition was determined. Of the various reasons that were considered to explain isoenzyme occurrence, a genetic basis was considered most likely. The enzyme was found to exhibit substrate inhibition with some substrates below pH 6, while above pH 8 it exhibited downwardly curving Lineweaver-Burk plots of the type that are generally described as "substrate activation". The observation of a phosphotransferase activity was consistent with the formation of a covalent phosphoenzyme intermediate, while inactivation by diethyl pyrocarbonate was consistent with the presence of an active site histidine.

Acid Phosphatase↗
PubMed1990

Drug binding by branched DNA: selective interaction of the dye stains-all with an immobile junction.

The thiacarbocyanine dye Stains-All (4,5:4',5'-dibenzo-3,3'-diethyl-9-methylthiacarbocyanine bromide) is one of a large number of cyanine dyes introduced as photosensitizers in the photographic industry. Stains-All is used in histology as a stain for nucleic acids, proteins, polysaccharides, and lipids. We report here that the dye colors branched DNA molecules differently from linear duplexes and use footprinting experiments with methidiumpropyl-EDTA-Fe(II) [MPE.Fe(II)] and bis(o-phenanthroline)copper(I) [(O-P)2Cu(I)] to show that Stains-All interacts preferentially at the branch point of a four-arm DNA structure. A titration experiment allows us to estimate that the interaction of the dye with the branch has a dissociation constant below 45 nM, tighter than that of ethidium or methidium by over 2 orders of magnitude. Probing the interaction with the purine-specific reagent diethyl pyrocarbonate (DEPC) implies that the dye induces an asymmetric distortion near the branch in the major grooves of double helix in the junction.

Absorption↗
PubMed1996

Biosynthesis of lipophosphoglycan from Leishmania major: solubilization and characterization of a (beta 1-3)-galactosyltransferase.

Lipophosphoglycan (LPG), is the major cell surface molecule of promastigotes of all Leishmania species. It is comprised of three domains: a conserved glycosylphosphatidylinositol anchor linked to a repeating phosphorylated disaccharide (P2; PO4-6Gal beta 1-4Man alpha 1-) backbone and capped with a neutral oligosaccharide. In Leishmania major the backbone is substituted at the C(O)3 of the Galp residue with side chains containing Galp, Glcp and Arap residues whereas in Leishmania donovani the backbone is unsubstituted. We report the solubilization of a (beta 1-3) galactosyltransferase [(beta 1-3)GalT] from a L. major microsomal preparation using Triton X-100. Solubilization occurs with a 10-fold stimulation of enzyme activity. This (beta 1-3)GalT specifically transfers Gal residues from UDP-Gal to exogenously added L. donovani LPG acceptor. Depolymerization of the [14C]Gal-labelled LPG product with mild acid and analysis by high-performance anion-exchange chromatography detected only the phosphotrisaccharide. (P3; PO4-6([14C]Gal beta 1-3-4Man alpha 1-) found in L. major LPG. This contrasts with the activity of the membrane-bound enzyme which also synthesizes the larger phosphosaccharide units[Ng, Handman and Bacic (1994) Glycobiology 4, 845-853]. This suggests that more than one (beta 1-3)GalT is involved in the addition of these Gal units and that the solubilized activity is the (beta 1-3)GalT that adds the first beta Gal residue to the acceptor. The (beta 1-3)GalT was partially purified by lectin-affinity chromatography and used to establish the K(m) values for UDP-Gal (445 microM) and L. donovani acceptor (280 microM as P2 molar equivalent) in kinetic assays. Inhibition studies with various glycosides and mono- and di-saccharides established the P2 repeating unit as the minimum acceptor structure recognized by (beta 1-3)GalT. The detergent-solubilized (beta 1-3)GalT was reversibly inactivated by millimolar concentrations of univalent anionic salts. The (beta 1-3)GalT had an absolute requirement for Mn2+ and also required Mg2+ for optimum activity; Mg2+ cannot substitute for Mn2+, which is loosely bound to beta (1-3)GalT and is probably involved in the correct folding of the enzyme. The (beta 1-3)GalT was unaffected by Ca2+ ions, but were irreversibly inactivated by micromolar levels of transition metal ions (Cu2+ > Zn2+ > Ni2 > Co2+). The (beta 1-3)GalT activity was also inhibited by diethyl pyrocarbonate, but not by N-ethylmaleimide or iodoacetamide, suggesting that active-site histidine residues, rather than cysteine residue(s), are important for enzyme activity.

Animals↗
PubMed1993

Histidine 296 is essential for the catalysis in Lactobacillus plantarum D-lactate dehydrogenase.

Two His residues, His-205 and His-296, in Lactobacillus plantarum D-lactate dehydrogenase are highly conserved in the D-isomer-specific 2-hydroxyacid dehydrogenase family, suggesting that they are candidates for the catalytic His in the enzyme. The substitution of His-296 with Tyr by means of site-directed mutagenesis induced a drastic decrease in the reaction rate, while a His-205 to Tyr substitution induced no large change in the catalytic properties. In pyruvate reduction, the Tyr-296 enzyme showed a slightly increased Km below pH 6.0 but no significant pH dependence above pH 6.0, where the wild-type enzyme showed an increased Km value. In D-lactate oxidation, in contrast, the Tyr-296 enzyme showed a greatly increased Km value for D-lactate and strong pH dependence. An additional substitution of His-296 with Gln induced more complete loss of the catalytic activity. In contrast to the Tyr-296 enzyme, the Gln-296 enzyme showed a greatly increased Km value and a strong pH-dependent reaction rate even in pyruvate reduction. Unlike the wild-type or His-205 enzyme, both the Tyr-296 and Gln-296 enzymes showed significant resistance against diethyl pyrocarbonate. These results clearly indicate that His-296 is essential for the catalysis by D-lactate dehydrogenase, as in the case of His-195 in L-lactate dehydrogenase.

Amino Acid Sequence↗
PubMed1995

Mutations within a highly conserved sequence present in the X region of phosphoinositide-specific phospholipase C-delta 1.

Phosphoinositide-specific phospholipase C (PI-PLC) enzymes have considerable structural similarity within limited regions (X and Y) implicated in catalysis. The role of residues contained within a highly conserved sequence present in the X region was investigated by site-directed mutagenesis of PLC-delta 1 isoenzyme. Seven residues (Ser-308, Ser-309, Ser-310, His-311, Thr-313, Tyr-314, and Gln-319) were individually replaced by alanine or glutamine (His-311). Replacement of two residues, His-311 and Tyr-314, resulted in a dramatic reduction of enzyme activity. The kcat of hydrolysis of phosphatidylinositol 4,5-bisphosphate by H311A and Y314A mutants was reduced 1000- and 10-fold respectively, with little effect on Km. Further analysis of H311A and Y314A mutants, using limited proteolysis and circular dichroism, had shown that no major structural alterations had occurred. Since site-directed mutagenesis demonstrated the importance of histidine residues, their role in enzyme function was also analysed by chemical modification with diethyl pyrocarbonate. This modification of histidine residues resulted in the reduction of enzyme activity and also indicated that more than one residue could be important.

Amino Acid Sequence↗
PubMed1976

Mechanism of pancreatic lipase action. 2. Catalytic properties of modified lipases.

Reaction of lipase with diethyl pyrocarbonate results in the modification of three histidine residues. One is highly reactive, although without affecting the activity, while the two others react more slowly with a concomitant loss of activity on both dissolved and emulsified substrates. As previously shown, lipase can also be modified either by reaction of five carboxyl groups with carbodiimide (5N-lipase) or by esterification of one serine residue with diethyl p-nitrophenyl phosphate (DP-lipase). In the three cases, the activity on emulsified substrates is abolished. The modification of histidine residues results also in a loss of activity on dissolved substrates, suggesting that the essential histidine is at (or close to) the active site. The ability of lipase to be adsorbed on siliconized glass beads is not impaired in this reaction. By contrast, 5N-lipase is still able to hydrolyze dissolved monomeric substrates and to adsorb on siliconized glass beands. Therefore, the essential carboxyl group is assumed to play an important role in the interfacial activation. Finally, since DP-lipase is still fully active on dissolved p-nitrophenyl acetate, the serine residue, which has been previously suggested to be the acylable one, is more likely implicated in the recognition and the binding to interfaces, as confirmed by the inability of DP-lipase to be adsorbed on siliconized glass beads.

Diethyl Pyrocarbonate↗
PubMed2000

Determination of transport kinetics of chick MCT3 monocarboxylate transporter from retinal pigment epithelium by expression in genetically modified yeast.

Monocarboxylate transporters (MCTs) comprise a group of highly homologous proteins that reside in the plasma membrane of almost all cells and which mediate the 1:1 electroneutral transport of a proton and a lactate ion. The isoform MCT3 is restricted to the basal membrane of the retinal pigment epithelium where it regulates lactate levels in the neural retina. Kinetic analysis of this transporter poses formidable difficulties due to the presence of multiple lactate transporters and their complex interaction with MCTs in adjacent cells. To circumvent these problems, we expressed both the MCT3 gene and a green fluorescent protein-tagged MCT3 construct in Saccharomyces cerevisiae. Since L-lactate metabolism in yeast depends on the CYB2 gene, we disrupted CYB2 to study the MCT3 transporter activity free from the complications of metabolism. Under these conditions L-lactate uptake varied inversely with pH, greater uptake being associated with lower pH. Whereas the V(max) was invariant, the K(m) increased severalfold as the pH rose from 6 to 8. In addition, MCT3 was highly resistant to a number of "classical" inhibitors of lactate transport. Last, studies with diethyl pyrocarbonate and p-chloromercuribenzenesulfonate set limitations on the locus of potential residues involved in the critical site of lactate translocation.

4-Chloromercuribenzenesulfonate↗
PubMed1985

Interaction between the mitochondrial ATP synthetase and ATPase inhibitor protein. Active/inactive slow pH-dependent transitions of the inhibitor protein.

The rate of mitochondrial ATPase inactivation by the naturally occurring inhibitor protein in the presence of saturating ATP and Mg2+ at pH 8.0 depends hyperbolically on the amount of inhibitor added; the upper limit of an apparent first-order constant for the inactivation process is 1.0(-1) at 25 degrees C. A dramatic difference in the inactivation rate is observed when the protein inhibitor is added to the same assay system from either acidic (pH 4.8) or alkaline (pH 8.2) solutions. The slow reversible transition of the inhibitor from its rapidly reacting 'acidic' form to the slow reacting 'alkaline' form occurs when the solution of the protein inhibitor is subjected to a pH-jump from 4.8 to 8.2 (t1/2 approximately 30s at 25 degrees C). The pH-profile of the inhibitor active/inactive equilibrium suggests that a group with pKa approximately 6.5 is involved in the transition. Treatment of the inhibitor protein with a histidine-specific reagent (e.g. diethyl pyrocarbonate) abolishes its inactivating effect on the ATPase activity. It is concluded that the protonation/deprotonation of the inhibitor protein followed by its slow conformational changes is the rate-limiting step in the inhibitor-ATP synthetase interaction.

Animals↗
PubMed1996

The isolation and some properties of the membrane-bound lactate dehydrogenase of Paracoccus denitrificans.

Membrane-bound lactate dehydrogenase was isolated from the cells of Paracoccus denitrificans by a combination of phase separation and chromatographic methods. It was NAD(+)-independent; phenazine methosulphate and 2,6-dichlorophenolindophenol could act as electron acceptors. It preferred D-lactate over the L-form (K(m) = 0.81 and 4.40 mM, respectively). Relative molecular weight estimations were 54000 +/- 3000 (electrophoresis) and 50000 +/- 5000 (gel chromatography) It was inhibited by thenoyltrifluoroacetone and diethyl pyrocarbonate, SH-reagents were without effect. No other lactate dehydrogenase activity was found in any subcellular fraction of cells grown on D,L-lactate, L-lactate, glucose or succinate.

Acetone↗
PubMed1985

Kinetic analysis of respiratory nitrate reductase from Escherichia coli K12.

Purified respiratory nitrate reductase from Escherichia coli is able to use either reduced viologen dyes or quinols as the electron donor and nitrate, chlorate, or bromate as the electron acceptor. When reduced viologen dyes act as the electron donor, the enzyme follows a compulsory-order, "Theorell-Chance" mechanism, in which it is an enzyme-nitrate complex that is reduced rather than the free enzyme. In contrast, if quinols are used as the electron donor, then the enzyme operates by a two-site, enzyme-substitution mechanism. Partial proteolysis of the cytochrome b containing holoenzyme by trypsin results in loss of cytochrome b and in cleavage of one of the enzyme's subunits. The cytochrome-free derivative exhibits a viologen dye dependent activity that is indistinguishable from that of the holoenzyme, but it is incapable of catalyzing the quinol-dependent reaction. The quinol-dependent, but not the viologen dye dependent, activity is inhibited irreversibly by exposure to diethyl pyrocarbonate and reversibly by treatment with 2-n-heptyl-4-hydroxyquinoline N-oxide. We conclude that the holoenzyme has two independent and spatially distinct active sites, one for quinol oxidation and the other for nitrate reduction.

Cytochrome b Group↗
PubMed1988

Chemical probing of homopurine-homopyrimidine mirror repeats in supercoiled DNA.

We have recently shown that under superhelical stress and/or acid pH the homopurine-homopyrimidine tracts conforming to the mirror symmetry (H palindromes) form a novel DNA structure, the H form. According to our model, the H form includes (1) a triplex formed by half of the purine strand and by the homopyrimidine hairpin and (2) the unstructured other half of the purine strand. We used four specially designed sequences to demonstrate that, in accordance with our model, the mirror symmetry is essential for facile formation of the H form detected by two-dimensional gel electrophoresis. Here we report that, under conditions favouring the H-form extrusion, guanines of the 3' half of the purine strand are protected against alkylation by dimethylsulphate, whereas adenines of the 5' half of the purine strand react with diethyl pyrocarbonate. These data indicate that the 3' half of the homopurine strand is within the triplex whereas the 5' half is unstructured, in full agreement with our model.

DNA, Superhelical↗