Evidence for histidine in the triethyltin-binding site of rat haemoglobin.
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ABSTRACT:
SUBMITTER: Rose MS
PROVIDER: S-EPMC1187799 | biostudies-other | 1969 Jan
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Triethyltin binding to cat haemoglobin was measured after pretreatment of the protein with diethyl pyrocarbonate at pH 6.0,iodoacetamide or phenylmercuric acetate or by photo-oxidation in the presence of Methylene Blue. The pentaco-ordinate nature of the binding of triethyltin to cat haemoglobin is confirmed by the inability of intramolecularly pentaco-ordinate tin compounds to compete. Consideration of the symmetry of the haemoglobin molecule in the light of the above results suggests that a unique arrangement of histidine and cysteine residues is required for the binding of triethyltin. The effects of treatment with diethyl pyrocarbonate of other preparations which bind triethyltin (rat liver supernatant, a fraction from rat liver mitochondria and rat brain myelin) were determined and shown to be complex.
| S-EPMC1186395 | biostudies-other
Project description:Triethyltin binds to native cat and rat haemoglobin but not to their denatured forms or to other animal haemoglobins. Two molecules of the organotin bind to one molecule of R-state cat haemoglobin with affinity constants of about 1 X 10(5) M-1. Little or no triethyltin is bound to the deoxygenated (T-state) protein. Binding appears to be dependent upon the existence of a specific three-dimensional configuration of cysteine and histidine residues. The properties of the triethyltin-cat haemoglobin complex are consistent with those of a haemoglobin conformer whose allosteric equilibrium is displaced toward the R-state. Its oxygen affinity and rate of oxidation by nitrite is increased, and the rate of reduction of the methaemoglobin derivative by ascorbate is decreased. These effects of triethyltin are opposite and antagonistic to the effects of inositol hexaphosphate. They are exerted on the alpha- as well as beta-haem groups, even though triethyltin is bound at sites on alpha-globin far removed from the haem groups.
| S-EPMC1153049 | biostudies-other
Project description:A protein fraction was isolated from guinea-pig liver that binds triethyltin with an affinity of approx. 2x10(6)m(-1) at pH8.0. It was shown that the protein responsible for binding 70% of the triethyltin found in guinea-pig liver after injection of radioactively labelled triethyltin is at most a few per cent of the total liver protein. Evidence is presented from the kinetics of loss of binding and loss of certain amino acids on photo-oxidation with either Methylene Blue or Rose Bengal that each binding site consists of two histidine residues.
| S-EPMC1179579 | biostudies-other
Project description:Cat haemoglobin binds 2 mol of triethyltin/mol of haemoglobin. Pretreatment of the haemoglobin with diethyl pyrocarbonate at pH6.0 prevents binding to one site only, whereas photo-oxidation with Methylene Blue removes both sites. Pretreatment of rat haemoglobin with diethyl pyrocarbonate also leads to the loss of one binding site. The possibility is discussed that the two binding sites for triethyltin on both cat and rat haemoglobin have a different chemical nature.
| S-EPMC1164739 | biostudies-other
Project description:The histidine ATP-binding cassette (ABC) transporter of Salmonella enterica serovar Typhimurium is among the best-studied type I ABC import systems. The transporter consists of two transmembrane subunits, HisQ and HisM, and a homodimer of the nucleotide-binding subunit, HisP. Substrates are delivered by two periplasmic solute binding proteins, HisJ and LAO, with preferences for histidine and for lysine, arginine, and ornithine, respectively. A homology model was built by using the arginine-bound crystal structure of the closely related Art(QN)2 transporter of Thermoanaerobacter tengcongensis as the template. In the homodimeric Art(QN)2, one substrate molecule is bound to each of the ArtQ subunits, whereas the structural model and sequence alignments predict only one substrate molecule in contact with HisM. To address the question whether one or two binding sites exist in heterodimeric HisQM, we have studied the functional consequences of mutations by monitoring (i) the complementation of growth on d-histidine of auxotrophic tester strains, (ii) the growth of tester strains on arginine as a nitrogen source, and (iii) ATPase activity of purified variants in a lipid environment. Our results demonstrate that two negatively charged residues, namely, HisM-E166 and HisQ-D61, are indispensable for function. Furthermore, the complete reconstruction of an ArtQ-like binding site in HisQ resulted in an inactive transporter. Likewise, switching the positions of both negatively charged residues between HisQ and HisM caused transport-deficient phenotypes. Thus, we propose that one substrate molecule is primarily liganded by residues of HisM while HisQ-D61 forms a crucial salt bridge with the ?-amino group of the substrate.IMPORTANCE Canonical ATP-binding cassette (ABC) importers are major players in the translocation of numerous nutrients, vitamins, and growth factors to the cytoplasm of prokaryotes. Moreover, some ABC importers have been identified as virulence factors in bacterial pathogenesis. Thus, a full understanding of their mode of action is considered a prerequisite, among others, for the development of novel antibacterial drugs. However, mainly owing to the lack of structural information, the knowledge of the chemical nature and number of substrate binding sites formed by the transmembrane subunits of ABC importers is scarce. Here, we provide evidence from mutational analyses that, in contrast to homologous homodimeric systems, the heterodimeric histidine transporter of Salmonella enterica serovar Typhimurium is liganding only one substrate molecule between its transmembrane subunits, HisM and HisQ.
| S-EPMC6304659 | biostudies-literature
Project description:Papain was irreversibly inhibited by 1,3-dibromoacetone, a reagent designed to react first with the active-site cysteine residue and subsequently with a second nucleophile. The molecular weight of the inhibited enzyme was indistinguishable from that of papain itself, and no evidence of dimeric or oligomeric species was found. The optical-rotatory-dispersion curves of chloroacetone-inhibited papain and 1,3-dibromoacetone-inhibited papain were essentially similar. Amino acid analysis of the 1,3-dibromo[2-(14)C]acetone-inhibited enzyme and the performic acid-oxidized material clearly showed that a cysteine and histidine residue had been alkylated through the thiol and N-1 of the imidazole group respectively. These groups must therefore be within 5å of each other in the tertiary structure of papain. Possible mechanistic implications are briefly discussed.
| S-EPMC1198892 | biostudies-other
Project description:A synthetic approach was employed to identify the haptoglobin-binding site on the alpha-chain of human haemoglobin. This approach cosists of the synthesis of a series of consecutive overlapping peptides that, together, systematically represent the entire protein chain. Fourteen peptides were synthesized (alpha 1-15, alpha 11-25, alpha 21-35, alpha 31-45, alpha 41-55, alpha 51-65, alpha 61-75, alpha 71-85, alpha 81-95, alpha 91-105, alpha 101-115, alpha 111-125, alpha 121-135 and alpha 131-141), and their ability bind human haptoglobin was studied, Only peptide alpha 121-135 bound haptoglobin significantly. On this basis we conclude that the haptoglobin-binding site on the alpha-chain of haemoglobin resides within, but does not necessarily encompass all of, the region alpha 121-135.
| S-EPMC1163152 | biostudies-other
Project description:Zn2+ is known to increase the 02 affinity of human haemoglobin. Previous data suggested that Zn2+ exerts its effect by directly binding to haemoglobin, rather than by competing with or binding to 2,3-bisphosphoglycerate. It was also shown that there are two 02-linked zinc-binding sites in haemoglobin, and that Zn2+ does not significantly alter haemoglobin co-operativity or the alkaline Bohr effect. The effect of Zn2+ on 02 affinity of haemoglobin can also be observed for other haemoglobins as diverse as those of cow and chicken. This paper presents new data on the haemoglobin-zinc interaction for normal haemoglobin, des-His146beta-haemoglobin and N-ethylsuccinimide-haemoglobin of humans. For normal haemoglobin (0.05 mM in tetramers), at 20 degrees C in buffer containing 0.1 M-Cl-, 02-dissociation-curve experiments showed that the addition of 0.4-0.5 mM-ZnS04 did not change the Bohr effect between pH 6.71 and 7.29. Similar experiments, with "zinc-ion buffers", showed that the value of the Hill coefficient, h, decreased only slightly if the concentration of free Zn2+ was held constant. For N-ethylsuccinimide-haemoglobin, Zn2+ caused less increase in O2 affinity than for normal haemoglobin. These studies, together with data on the equilibrium binding of Zn2+ to oxy-, deoxy- and des-His146beta-haemoglobins, suggest that zinc is chelated in oxyhaemoglobin by at least three amino acids, two of which are histidine-146beta and cysteine-93beta.
| S-EPMC1183835 | biostudies-other
Project description:1. Yeast alcohol dehydrogenase (EC 1.1.1.1) is inhibited by stoicheiometric concentrations of diethyl pyrocarbonate. The inhibition is due to the acylation of a single histidine residue/monomer (mol.wt. 36000). 2. Alcohol dehydrogenase is also inhibited by stoicheiometric amounts of 5,5'-dithiobis-(2-nitrobenzoate), owing to the modification of a single cysteine residue/monomer. 3. Native alcohol dehydrogenase binds two molecules of reduced coenzyme/molecule of enzyme (mol.wt. 144000). 4. Modification of a single histidine residue/monomer by treatment with diethyl pyrocarbonate prevents the binding of acetamide in the ternary complex, enzyme-NADH-acetamede, but does not prevent the binding of NADH to the enzyme. 5. Modification of a single cysteine residue/monomer does not prevent the binding of acetamide to the ternary complex. After the modification of two thiol groups/monomer by treatment with 5,5'-dithiobis-(2-nitrobenzoate), the capacity of enzyme to bind coenzyme in the ternary complex was virtually abolished. 6. From the results presented in this paper we conclude that at least one histidine and one cysteine residue are closely associated in the substrate-binding site of alcohol dehydrogenase.
| S-EPMC1165260 | biostudies-other
Project description:Acyltransferases determine which extender units are incorporated into polyketide and fatty acid products. The ping-pong acyltransferase mechanism utilizes a serine in a conserved GHSxG motif. However, the role of the conserved histidine in this motif is poorly understood. We observed that a histidine to alanine mutation (H640A) in the GHSxG motif of the malonyl-CoA specific yersiniabactin acyltransferase results in an approximately seven-fold higher hydrolysis rate over the wildtype enzyme, while retaining transacylation activity. We propose two possibilities for the reduction in hydrolysis rate: either H640 structurally stabilizes the protein by hydrogen bonding with a conserved asparagine in the ferredoxin-like subdomain of the protein, or a water-mediated hydrogen bond between H640 and the malonyl moiety stabilizes the malonyl-O-AT ester intermediate.
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