Reactivity of sulphydryl groups of cytosolic and mitochondrial bovine aspartate aminotransferases

Summary Reactivity of sulphydryl groups of cytosolic and mitochondrial aspartate aminotransferases from ox heart has been studied. A total of 5 and 7 cysteine residues per monomer are present in cAATo and mAATo, respectively. In native conditions only a single sulphydryl group can be titrated by Nbs₂ while the catalytic activity remains unchanged, however in the mitochondrial isozyme the reactivity depends on the functional state of the enzyme. Reactivity toward NEM reveals the existence of a syncatalytic sulphydryl group in the cytosolic isozyme. Titration of cAATo with pMB at pH 8 and pH 5 confirms the existence of two exposed sulphydryl groups with a different reactivity. The results compared with those reported on the corresponding isozymes from pig and chicken heart show that syncatalytic sulphydryl groups are of general occurrence in these enzymes.     

Studies on 3-deoxy-d-arabinoheptulosonate-7-phosphate synthetase(phe) from escherichia coli k12. 3. Structural studies.

1. Investigations with structural analogues of phenylalanine indicated an absolute requirement for the aromatic ring and both the alpha-carboxyl and alpha-amino groups of phenylalanine for inhibition of 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase(phe) activity. Replacement of the alpha-H atom with a methyl group does not decrease the inhibition greatly. Varying degrees of inhibition were observed with o, m and p mono-substituted fluoro, chloro and hydroxy phenylalanines. D-Phenylalanine and several metabolites of the aromatic biosynthetic pathways do not inhibit enzymic activity. 2. Circular dichroism studies indicated that the native enzyme possesses approximately 26% alpha-helix. Both circular dichroic and ultraviolet difference spectra indicated that the addition of phenylalanine to the synthetase induces a conformational change involving a small alteration of the secondary structure and large alterations in th interactions of some of the aromatic residues of the enzyme. In particular, a tryptophan residue moves from an extremly hydrophobic environment to one less hydrophobic. 3. Kd for the binding of phenylalanine to the enzyme was determined spectrophotometrically to be 75 muM. 4. Chemical modification studies suggested that a sulphydryl group and possibly a lysine residue may be implicated in the catalytic activity of the enzyme.     

Alternate Positions for the Sulphydryl Group in β-Lactoglobulin: the Significance for Sulphydryl Location in Other Proteins

EARLIER studies of the location of the single cysteine residue and the two disulphide bridges in bovine β-lactoglobulins A and B¹, for each of which the monomer is a single chain of 162 residues and 18,000 molecular weight^2,3, led to the conclusion that the sulphydryl group is at position 69 and that the disulphides bridge positions 123 to 160 and 57 to 70. These results were based on diagonal peptide studies⁴ and on the composition of peptides in which the sulphydryl group had been labelled with ¹⁴C-iodoacetamide, the disulphide bridges being left intact. Use was made of the partial amino-acid sequence given by Frank and Braunitzer⁵ and the reasonable assumption was made that the sulphydryl occurred in only one position. Subsequently, Shaw⁶ has shown that the sequence of Frank and Braunitzer⁵ showing Cys residues adjacent at positions 69 and 70 is incorrect and that they are separated by a glutamine, the sequence for positions 67 to 71 for the Bvariant being Ala.Cys.Gln.Cys.Leu. Autoradiography of the dansyl amino-acid derivatives formed during the sequence determination of this pentapeptide indicated that both residues 68 and 70 seemed to have been labelled and so we have given further consideration to the sulphydryl location. It has been found that although it does occur at 68, with 57 and 70 disulphide bridged, there is also an equal amount of protein present with the sulphydryl at 70, with 57 and 68 disulphide bridged. We discuss this additional finding here and the significance for the determination of the location of sulphydryl groups in other proteins.     

Characterization of Mycobacterium tuberculosis diaminopimelic acid epimerase: paired cysteine residues are crucial for racemization

Recently, the overproduction of Mycobacterium tuberculosis diaminopimelic acid (DAP) epimerase MtDapF in Escherichia coli using a novel codon alteration cloning strategy and the characterization of the purified enzyme was reported. In the present study, the effect of sulphydryl alkylating agents on the in vitro activity of M. tuberculosis DapF was tested. The complete inhibition of the enzyme by 2-nitro-5-thiocyanatobenzoate, 5,5'-dithio-bis(2-nitrobenzoic acid) and 1,2-benzisothiazolidine-3-one at nanomolar concentrations suggested that these sulphydryl alkylating agents modify functionally significant cysteine residues at or near the active site of the epimerase. Consequently, the authors extended the characterization of MtDapF by studying the role of the two strictly conserved cysteine residues. The putative catalytic residues Cys87 and Cys226 of MtDapF were replaced individually with both serine and alanine. Residual epimerase activity was detected for both the serine replacement mutants C87S and C226S in vitro. Kinetic analyses revealed that, despite a decrease in the K(M) value of the C87S mutant for DAP that presumably indicates an increase in nonproductive substrate binding, the catalytic efficiency of both serine substitution mutants was severely compromised. When either C87 or C226 were substituted with alanine, epimerase activity was not detected emphasizing the importance of both of these cysteine residues in catalysis.     

Isatin-enzyme interactions. VI. Inhibition of rat kidney alkaline phosphatase.]

Interaction of isatin with rat kidney alkaline phosphatase has been studied. Mode of attachment of isatin with the enzyme protein is most likely through amino group(s), which is also imperative for catalysis. Sulphydryl group(s) do not seem to be involved in enzyme action. Zinc is also needed for enzyme activity. Use of sulphydryl compounds suggests that isatin inhibition of the enzyme is through attachment at the metal site. However, this inhibition may not only be due to simple chelation of the metal by isatin.     

Localization and primary characterization of bile salt hydrolase from Lactobacillus reuteri

The bile salt hydrolase (BSH) of Lactobacillus reuteri CRL 1098 is a single, constitutive, intracellular enzyme which is only detectable in stationary phase cells. It has optimal activity at pH 4.5–5.5 and 37–45 °C. The enzyme (80 kDa apparent mass) has sulphydryl groups in the catalytic active site and hydrolyzes both glycine and taurine conjugated bile acids with higher affinity for glyco-conjugates.     

Enzymic synthesis of steroid sulphates. XI. Study of the oestrogen binding site of oestrogen sulphotransferase by affinity labelling with 4-mercuri-17beta-oestradiol.

Oistrogen sulphotransferase (3"-phosphoadenylylsulphate: oestrone sulphotransferase, EC 2.8.2.4) contains asingle sulphydryl group thought to be at, or near, the oestrogen-binding site. 4-mercuri-17beta-oestradiol, the activity of the enzyme decreased with increasing concentration of the oestrogen derivative. However, some 40% of the activity remained when all the sulphydryl had reacted to form mercaptide. Formation of mercaptide was only marginally decreased in the presence of the substrate 17beta-oestradiol. Other steroids, such as 11-deoxycorticosterone and testosterone, which are non-substrates for the enzyme, were more effective than 17beta-oestradiol in inhibiting mercaptide formation. Bovine serum albumin also reacted with 4-mercure-17beta-oestradiol and the effects of various steroids on mercaptide formation by the affinity label closely paralleled those found for the enzyme. 2t is concluded that the single sulphydryl group in the enzyme is not directly involved in the binding of oestrogen at the active site but is perhaps in closer proximity to a second site capable of binding certain non-substrate steroids.     

Purification and kinetic properties of chalcone-flavanone isomerase from soya bean.

The chalcone-flavanone isomerase from soya bean seed has been purified 8300-fold. A molecular weight of 15,600 plus or minus 1000 has been determined for the enzyme. Effects of iodoacetamide and sodium tetrathionate on the enzyme, and pH dependence of the catalytic step, indicate that a sulphydryl group is not involved in the reaction mechanism and the catalytic group is probably an imidazole side chain in the basic form. The kinetics of the isomerisation of isoliquiritigenin to liquiritigenin have been examined and show that at pH 7.6 the reaction is reversible with an equilibrium constant of 37 in favour of flavanone. A number of flavonoid compounds competitively inhibit the reaction, suggesting a possible regulatory role for this enzyme in flavonoid biosynthesis.     

Exposed sulphydryl groups of chicken haemoglobins: globin localization and effect of oxygenation on their reactivity

The number and the reactivity of the sulphydryl groups of the two major haemoglobin fractions of adult fowl erythrocytes, Hb-1 and Hb-2², have been determined with paramereuribenzoate and iodoacetamide. The number of sulphydryl groups of Hb-1 that react with paramereuribenzoate and their kinetics of combination are dependent on the ligand state of the molecule. Experiments with [¹⁴C]iodoacetamide show that two sulphydryl groups of the β chains are always reactive, though with different kinetics. One sulphydryl group appears reactive only with paramereuribenzoate and only when the molecule is oxygenated.The number of reactive sulphydryl groups of Hb-2 does not change with the ligand state of the molecule but the kinetics of combination is slower for the deoxy form. Reaction with [¹⁴C]iodoaoetamide shows that each α chain has one fast-reacting sulphydryl group and eachβ chain has one fast and one slowreacting sulphydryl group. The fast-reacting groups of Hb-2 can be blocked selectively with iodoacetamide. Tentative identification of the reacting sulphydryl groups of Hb-2 has been made on the basis of their corresponding positions in human haemoglobin.     

The location of sulphydryl groups in alpha-crystallin

The microenvironments of the sulphydryl groups in the multimeric protein, alpha-crystallin, were studied by examining: the rate of the reaction of the groups with DTNB; the effect of increasing urea concentrations on their accessibilities; and the quenching of a fluorescent probe. In foetal bovine alpha-crystallin (1 SH/alpha A subunit) both kinetic and quenching studies indicated that over 90% of the sulphydryl groups fell into a single buried class; the remainder was exposed. In the human protein (2 SH/alpha A subunit), half of the groups were buried and the other half exposed. Accessible sulphydryl groups increased gradually as the urea concentration was increased, with complete exposure at about 4.0 M. Sedimentation velocity analyses revealed that no significant dissociation of the aggregates into subunits occurred below 3.5 M urea, at which point over 80% of the sulphydryl groups were exposed. An age-dependent increase (3-35%) was found in the proportion of exposed sulphydryl groups in bovine alpha-crystallin and a decrease in the urea concentration required to expose the remainder. It was concluded that the single cysteine is buried in the newly synthesized protein, but becomes solvent-exposed as a result of age-related conformational changes. Our observations are consistent with a quaternary structure in which all alpha A subunits occupy equivalent sites.     

Effects of metals on enzyme activity in plants

Abstract. Uptake of phytotoxic amounts of metal by higher plants or algae can result in inhibition of several enzymes, and in increase in activity (= induction) of others. Two mechanisms of enzyme inhibition predominate: (1) binding of the metal to sulphydryl groups, involved in the catalytic actionor structural integrity of enzymes, and (2) deficiency of an essential metal in metalloproteins or metal-protein complexes, eventually combined with substitution of the toxic metal for the deficient element. Metal accumulation in the cellular compartment of the enzyme is a prerequisite for enzyme inhibition in vivo. The induction of some enzymes is considered to play a significant role in the stress metabolism, induced by metal phytotoxicity. Peroxidase induction is likely to be related to oxidative reactions at the biomembrane; several enzymes of the intermediary metabolism might be stimulated to compensate for metal-sensitive photosynthetic reactions. The induction of enzymes and metal-specific changes in isoperoxidase pattern can be used as diagnostic criteria to evaluate the phytotoxicity of soils, contaminated by several metals. Lines for future research on metal phytotoxicity are proposed, involving the study of inhibition and induction of enzymes at the different cell membranes (especially the plasmamembrane) in vivo.     

Inhibition of platelet behaviour by feverfew: a mechanism of action involving sulphydryl groups

Extracts of feverfew inhibit platelet aggregation and the platelet release reaction. The active components are believed to be sesquiterpene lactones such as parthenolide. Evidence is presented that inhibition of platelet behaviour is via neutralization of sulphydryl groups either inside or outside the cell. The precise nature of the sulphydryl groups that are susceptible to feverfew and are involved in platelet aggregation and the release reaction have not yet been defined.     

An S-adenosyl-l-methionine; coniine methyltransferase from Conium maculatum

Evidence is presented for a cell free system from Conium maculatum which catalyses the transfer of a methyl group from S-adenoysl-l-methionine to coniine with the formation of N-methyl coniine. Maximum enzyme activity which occurred in the unripe fruits was enhanced by dithiothreitol, and evidence for the role of sulphydryl groups of the enzyme was obtained from inhibition with p-CMB, iodoacetamide and N-methyl maleimide. A divalent metal cation dependency was not detected.     

Sulphydryl groups in photosynthetic energy conservation. V. Localization of the new disulfide bridges formed by o-iodosobenzoate in coupling factor of spinach chloroplasts.

1. Chemical modification by o-iodosobenzoate of soluble chloroplast coupling factor 1 (CF1) during heat activation resulted in inhibition of its Ca-ATPase activity and in the formation of two new intrapeptide disulfide bridges as suggested by: (a) the disappearance of three out of four accessible thiol groups, two from gamma and one from a beta subunit as a consequence of CF1 modification by o-iodosobenzoate; (b) the total free sulphydryl groups of CF1 were reduced from 8 to 4 after modification of CF1 by o-iodosobenzoate. Two groups disappeared from beta and two from gamma subunits; (c) a second heating step of CF1 in the presence of 10 mM dithioerythritol reversed the inhibition of the ATPase and reduced both the newly formed disulfide bridges and those present in native CF1. 2. Modification of chloroplasts in the light with o-iodosobenzoate resulted in the inhibition of photophosphorylation and ATPase. CF1 isolated and purified from these chloroplasts had its Ca-ATPase activity inhibited and two new disulfide bridges. The total number of free sulphydryl groups was reduced from 8 to 4 and three accessible groups disappeared from beta and gamma subunits.     

Ionizable groups linked to the reaction of 2,2'-dithiobispyridine with hemoglobin.

The pH-dependence of the second-order rate-constant for the reaction of 2,2'-dithiobispyridine with the CysF9(93) beta sulphydryl group of hemoglobin in the R quaternary structure is analyzed in terms of a tentative model based on the observation that this sulphydryl exists as a mixture of two tertiary conformations in dynamic equilibrium. For the four aquomethemoglobins studied (human A and S, dog and rabbit), the equation derived from this model gives a better fit than a simpler equation based on the assumption of only one tertiary conformation. For the corresponding carbonmonoxyhemoglobins the simpler equation gives a better fit. The dog and rabbit oxy and azidomet data are better fitted by the model equation, whereas the data for the corresponding human A and S derivatives are better fitted by the simpler equation. From the analysis pKa values of 6.1 and 8.7 are obtained for the ionization of groups coupled to the presumed conformational transition. The pKa of 6.1 is assigned to HisHC3(146) beta; the pKa of 8.7 is assigned to the CysF9(93) beta sulphydryl group in its external conformation. It is estimated that the pKa of this sulphydryl may be as high as 12.9 in its internal conformation.     

Isolation of stimulatory modulator from rat brain and its specific effect on guanosine 3':5'-monophosphate-dependent protein kinase from cerebellum and other tissues.

Mimicry of selective cytochalasin A (CA) inhibition of cellulase synthesis in the water mold $\text{Achlya}$ by the non-penetrating thiol reagent, p-chloromercuribenzene sulphonate suggests that CA may act as a non-permeant sulphydryl reagent, and that inhibition of cellulase synthesis may be exerted via relatively superficial thiol groups in the plasma membrane.     

Ornithine transaminase from Cucurbita maxima cotyledons

During germination a marked increase in both soluble and particulate ornthine transaminase occurs in pumpkin cotyledons. Both enzymes had a pH optimum of 8.3 and a requirement for ornthine and α-ketoglutarate. Other keto acids or amino donors showed little activity. The enzymes required an active sulphydryl group for maximum activity. Exogenous pyridoxal phosphate was not required, but hydroxylamine inhibited the reaction and added pyridoxal phosphate overcame this inhibition. Proline inhibited the reaction and may play a role in the fate of ornithine in pumpkin cotyledons.     

Inhibition of the activity of membrane-bound Ca2+-ATPase in the sarcoplasmic reticulum fragments of rabbit skeletal muscles by the alkaloid sanguinarine

Sanguinarine, a plant DNA-intercalator, is shown to inhibit the enzyme activity of the membrane-bound Ca2+-ATPase of rabbit skeletal muscle sarcoplasmic reticulum fragments. This inhibition could be interpreted by the well known ability of this alkaloid to interact with sulphydryl groups of the enzymes. Sanguinarine is a weaker inhibitor of this reaction than a sulphydryl group poison Ag+. The I50 is 3.10(-6) M for Ag+ and 7.10(-5) M for sanguinarine in the reaction medium with NO3- substituted for Cl-. In the standard reaction medium containing Cl-, the I50 for sanguinarine is 1.8.10(-4) M. In this case sanguinarine activates Ca2+-ATPase at low concentrations presumably because of uncoupling ATP hydrolysis from Ca2+ transport through membrane. Other agents studied are: DNA-intercalators--ethidium bromide, acriflavine, acridine orange; DNA-complexing antibiotics--actinomycin D, and olivomycin, alkaloids, quinine, morphine, berberine and an uncoupler of oxidative phosphorylation 2,4-dinitrophenol. These were found not to inhibit Ca2+-ATPase activity up to the concentrations of 10(-3)-10(-4) M.     

Human placental calcium activated neutral proteinase: Separation and functional characterization of subunits

The subunits of human placental milli calcium activated neutral proteinase and micro calcium activated neutral proteinase have been separated by partial denaturation with urea followed by molecular sieving, with a recovery of 82–91% of activity. The separated subunits were homogeneous, as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Their molecular sizes, catalytic activities and sulphydryl contents suggest that both the subunits of these two calcium activated neutral proteinases are distinct. The subunits were highly specific and could not be interchanged. Both the subunits of micro calcium activated neutral proteinase were catalytically active whereas only the 80 k subunit of milli calcium activated neutral proteinase was active. 30 k subunit of milli calcium activated neutral proteinase has a regulatory role since maximum activity of the 80 k subunit was elicited only in its presence. Activity of the reassociated subunits indicated that interaction is essential for the expression of optimum activity. Interaction of subunits rendered the enzymes less susceptible to inhibition by endogenous calcium activated neutral proteinase inhibitor.     

Inhibition of the catalytic subunit of cAMP-dependent protein kinase by dicyclohexylcarbodiimide

The hydrophobic carbodiimide dicyclohexylcarbodiimide (DCCD) has been shown to inhibit the catalytic (C) subunit of adenosine cyclic 3',5'-phosphate dependent protein kinase (EC 2.7.1.3) in a time-dependent, irreversible manner. The rate of inactivation was first order and showed saturation kinetics with an apparent Ki of 60 microM. Magnesium adenosine 5'-triphosphate (MgATP) was capable of protecting against this inhibition, whereas neither a synthetic peptide substrate nor histone afforded protection. Mg alone afforded some protection. When the catalytic subunit was aggregated with the regulatory subunit in the holoenzyme complex, no inhibition was observed. The inhibition was enhanced at low pH, suggesting that a carboxylic acid group was the target for interaction with DCCD. On the basis of the protection studies, it is most likely that this carboxylic acid group is associated with the MgATP binding site, perhaps serving as a ligand for the metal. Efforts to identify the site that was modified by DCCD included (1) modification with [14C]DCCD, (2) modification by DCCD in the presence of [3H]aniline, and (3) modification with DCCD and [14C]glycine ethyl ester. In no case was radioactivity incorporated into the protein, suggesting that the irreversible inhibition was due to an intramolecular cross-link between a reactive carboxylic acid group and a nearby amino group. Differential peptide mapping identified a single peptide that was consistently lost as a consequence of DCCD inhibition. This peptide (residues 166-189) contained four carboxylic acid residues as well as an internal Lys.(ABSTRACT TRUNCATED AT 250 WORDS)