共查询到20条相似文献,搜索用时 15 毫秒
1.
Yanet Fuentes-Martínez Felipe Medrano Anatoly K. Yatsimirsky 《Bioorganic chemistry》2010,38(4):173-2949
Protonation of an aminoglycoside antibiotic kanamycin A sulfate was studied by potentiometric titrations at variable ionic strength, sulfate concentration and temperature. From these results the association constants of differently protonated forms of kanamycin A with sulfate and enthalpy changes for protonation of each amino group were determined. The protonation of all amino groups of kanamycin A is exothermic, but the protonation enthalpy does not correlate with basicity as in a case of simple polyamines. The sites of stepwise protonation of kanamycin A have been assigned by analysis of 1H-13C-HSQC spectra at variable pH in D2O. Plots of chemical shifts for each H and C atom of kanamycin A vs. pH were fitted to the theoretical equation relating them to pKa values of ionogenic groups and it was observed that changes in chemical shifts of all atoms in ring C were controlled by ionization of a single amino group with pKa 7.98, in ring B by ionization of two amino groups with pKa 6.61 and 8.54, but in ring A all atoms felt ionization of one group with pKa 9.19 and some atoms felt ionization of a second group with pKa 6.51, which therefore should belong to amino group at C3 in ring B positioned closer to the ring A while higher pKa 8.54 can be assigned to the group at C1. This resolves the previously existed uncertainty in assignment of protonation sites in rings B and C. 相似文献
2.
Ribonuclease A has been trinitrophenylated to varying degrees by reaction with trinitrobenzenesulfonic acid. The reactive amino groups were identified by use of the peptides obtained from the oxidized TNP-RNase by tryptic and chymotryptic hydrolysis. From a quantitative study of the TNP-peptides it was possible to associate each amino group with values of pKma. It was shown that the lys-41 amino group had a pKa of 9.03 in TEA buffer. The pKa values of all of the other amino groups were dependent on the nature of the buffer (triethanolamine and phosphate) and on the pH. 相似文献
3.
We expand the spectroscopic utility of a well-known infrared and fluorescence probe, p-cyanophenylalanine, by showing that it can also serve as a pH sensor. This new application is based on the notion that the fluorescence quantum yield of this unnatural amino acid, when placed at or near the N-terminal end of a polypeptide, depends on the protonation status of the N-terminal amino group of the peptide. Using this pH sensor, we are able to determine the N-terminal pKa values of nine tripeptides and also the membrane penetration kinetics of a cell-penetrating peptide. Taken together, these examples demonstrate the applicability of using this unnatural amino acid fluorophore to study pH-dependent biological processes or events that accompany a pH change. 相似文献
4.
Spencer E. Taylor Erwin Buncel Albert R. Norris 《Journal of inorganic biochemistry》1981,15(2):131-141
Proton nmr spectroscopic evidence is presented for methylmercury(II) binding to the deprotonated amino groups in adenosine, 9-methyladenine, guanosine, 1-methylguanosine, and cytidine under basic conditions. Except for the guanosine case, 1H nmr spectra of the products from aqueous or ethanolic 1:1 mixtures of substrate and MeHgOH are consistent with methylmercuration of the deprotonated amino groups. Guanosine undergoes initial binding of MeHg to N1, and a second equivalent of MeHgOH is necessary to effect amino binding. The nmr spectra of the complexed adenine derivatives suggest that different geometrical isomers exist in (CD3)2SO solution, reflecting the partial double bond character of the C6N bond in these systems. Using a correlation relating the magnitude of the 199Hg-1H coupling constant (J) for MeHg-ligand complexes with the ligand pKa (J = ?3.88 pKa + 248.5, extending over 13 pK units, based on a variety of N and O donor ligands), estimates (± 0.3 pK unit) of the pKas of the amino groups of the above substrates have been made. In this way, pKa values of 15.5 (cytidine), 17.0 (adenosine and 9-methyladenine), 15.1 (guanosine), and 14.9 (1-methylguanosine) are obtained. In the cases where comparisons with literature pKa data can be made, good agreement is found. 相似文献
5.
Paulo Boschcov Wolfgang Seidel José Muradian Mineko Tominaga Antonio C.M. Paiva Luiz Juliano 《Bioorganic chemistry》1983,12(1):34-44
The pKa values for the proton dissociation of carboxyl, imidazolium, and ammonium groups for histidine and ten of its derivatives were determined electrometrically at seven temperatures in the range 10–40°C. The ΔH and ΔS values were estimated from the temperature dependence of the dissociation constants of histidine and its derivatives. These results and the pKa values compared in terms of inductive effect suggest an ion-dipole interaction between the protonated amino group and the unprotonated imidazole ring. The charge and the solvation effects of the neighboring groups are the main factors that determine the imidazole group pKa in histidine and its studied derivatives. The Nτ-H tautomer is favored over the Nπ-H by 1.6 kcal/mol, indicating that the inductive substituent effect at position 4 of the imidazole ring is the major component in determining this tautomeric preference. 相似文献
6.
Small reversible changes in the absorption spectra of HCN, CO, NO and O2 complexes of ferrous diacetyldeuteroperoxidase A, not hitherto observed, were attributed to proton dissociation of a distal amino acid residue. From spectrophotometric titration data the pKa was measured as 5.5 (HCN), 5.6 (ligand free), 6.0 (CO), 6.55 (NO) and 8.0 (O2). The value of 8.0 for the pKa of the O2 complex was also obtained from a curve of pH dependence of proton uptake in the reaction of the ferrous enzyme with O2. Absorption bands in the visible region were shifted to longer wavelengths in the order of CO to NO to O2 which is the decreasing order of the energy of π1 level of these diatomic ligands.The pKa values for CO complexes of ferroperoxidases, isoenzymes A and (B+C) were varied with substituents at the 2 and 4 positions of deuterohemin IX, and the ratio was about 0.3 in both series of isoenzyme preparations, where pK3 is a measure of basicity of pyrrole nitrogen.The present data support the previous conclusion (Yamada and Yamazaki (1974) Arch. Biochem. Biophys., 165, 728) that the pKa for ferroperoxidases, measured from small reversible changes in the absorption spectra, represents a proton dissociation constant of a distal amino acid residue and that there is hydrogen bonding between the residue and a ligand atom directly bound to the iron atom. 相似文献
7.
The uptake of glutamate in nerve synapses is carried out by the excitatory amino acid transporters (EAATs), involving the cotransport of a proton and three Na+ ions and the countertransport of a K+ ion. In this study, we use an EAAT3 homology model to calculate the pKa of several titratable residues around the glutamate binding site to locate the proton carrier site involved in the translocation of the substrate. After identifying E374 as the main candidate for carrying the proton, we calculate the protonation state of this residue in different conformations of EAAT3 and with different ligands bound. We find that E374 is protonated in the fully bound state, but removing the Na2 ion and the substrate reduces the pKa of this residue and favors the release of the proton to solution. Removing the remaining Na+ ions again favors the protonation of E374 in both the outward- and inward-facing states, hence the proton is not released in the empty transporter. By calculating the pKa of E374 with a K+ ion bound in three possible sites, we show that binding of the K+ ion is necessary for the release of the proton in the inward-facing state. This suggests a mechanism in which a K+ ion replaces one of the ligands bound to the transporter, which may explain the faster transport rates of the EAATs compared to its archaeal homologs. 相似文献
8.
The uptake of glutamate in nerve synapses is carried out by the excitatory amino acid transporters (EAATs), involving the cotransport of a proton and three Na+ ions and the countertransport of a K+ ion. In this study, we use an EAAT3 homology model to calculate the pKa of several titratable residues around the glutamate binding site to locate the proton carrier site involved in the translocation of the substrate. After identifying E374 as the main candidate for carrying the proton, we calculate the protonation state of this residue in different conformations of EAAT3 and with different ligands bound. We find that E374 is protonated in the fully bound state, but removing the Na2 ion and the substrate reduces the pKa of this residue and favors the release of the proton to solution. Removing the remaining Na+ ions again favors the protonation of E374 in both the outward- and inward-facing states, hence the proton is not released in the empty transporter. By calculating the pKa of E374 with a K+ ion bound in three possible sites, we show that binding of the K+ ion is necessary for the release of the proton in the inward-facing state. This suggests a mechanism in which a K+ ion replaces one of the ligands bound to the transporter, which may explain the faster transport rates of the EAATs compared to its archaeal homologs. 相似文献
9.
《Phytochemistry》1987,26(5):1299-1300
The effect ofpH on Km and Vmax values of coconut α-galactosidase indicates the involvement of two ionizing groups with pKa values of 3.5 and 6.5 in catalysis. Chemical modification has indicated the presence of two carboxyl groups, a tryptophan and a tyrosine, at or near the active site of α-galactosidase. Based on these facts a new mechanism of action for α-galactosidase is proposed in which the ionizing group with a pKa of 3.5 is a carboxyl group involved in stabilizing a carbonium ion intermediate and the ionizing group with a pKa of 6.5 is a carboxyl group perturbed due to the presence of a hydrophobic residues in its vicinity which donates a H+ ion in catalysis. 相似文献
10.
《Journal of inorganic biochemistry》1986,26(2):117-125
Myoglobin of Aplysia brasiliana (MbApB) has been recently purified and characterized and it was shown that the amino acid content is quite different from other myoglobins. A large number of aromatic residues was observed together with the existence of a unique histidine at the proximal heme position. Because of the numerous differences in the amino acid sequence between MbApB and whale myoglobin, it was interesting to investigate the interaction of metal ions like Cu2+ and Mn2+ with MbApB. In the present work Cu2+ complexes with Met-MbApB were studied and show a pH transition between different forms of coordination as revealed by EPR measurements. At high pH the EPR spectrum shows the coordination of the metal to at least four nitrogens from ϵ-NH3 lysine residues. At lower pH in the range 6.0–9.0 the copper binding site shows a pK change of some of the residues involved in metal coordination. Addition of one equivalent Cu2+ per protein does not alter the iron EPR signal. The manganese ion has one binding site in MbApB and a binding constant Ka = ( 11.5 ± 0.8) 103M−1. The binding of Cu2+ to MbApB is stronger than Mn2+, KaCu2+ >KaMn2+. 相似文献
11.
《Enzyme and microbial technology》2006,38(1-2):246-252
To study the effect of extraction protocols on extracellular polymeric substances (EPS) metal binding ability, EPS from two activated sludges were extracted by eight extraction protocols: three chemical treatments, four physical treatments and a control. Two pKa, for each EPS, were determined: pKa1 may be specific for carboxyl and phosphoric and pKa2 may be attributed to phenolic and amino functional groups according to EPS composition and IR spectra. EPS pKa values could be affected by the presence of extraction reagents and/or the modifications of EPS by extraction reagents.Complexation study performed at pH 7 by a polarographic method has always showed a greater affinity of EPS for Pb2+ than for Cd2+. The complexation properties of EPS extracted by chemical methods were greatly modified. Concerning EPS extracted by physical methods, their complexation properties were close except for EPS obtained by heating. Standardized extraction methods must be established as a function of the aims of the EPS study. 相似文献
12.
The potentiometric acid-base titration curve of fully protonated lysozyme at ionic strengths of 0.10 and 1.0 m has been performed. The stoichiometry and the pKa values of each titratable group have been determined through the linearization of titration curves. Two types of carboxylic groups with pKa values of 3.76 and 5.02, the imidazole group with pKa 7.37 and the amine group with pKa 9.63, have been identified at an ionic strength of 0.10 m at 25.0°C. The number of titratable groups found per mole of protein has been 5.12 and 5.60 for the two types of carboxylic groups, 1.13 for the imidazole group, and 3.19 for the amino groups. The endpoint of the titration of the protein obtained by this method accords quite well with the endpoint obtained by the use of Gran function applied to the excess of strong base. 相似文献
13.
Michael J. Harms Jamie L. Schlessman Gloria R. Sue Brian R. Cannon 《Journal of molecular biology》2009,389(1):34-299
The pKa values of internal ionizable groups are usually very different from the normal pKa values of ionizable groups in water. To examine the molecular determinants of pKa values of internal groups, we compared the properties of Lys, Asp, and Glu at internal position 38 in staphylococcal nuclease. Lys38 titrates with a normal or elevated pKa, whereas Asp38 and Glu38 titrate with elevated pKa values of 7.0 and 7.2, respectively. In the structure of the L38K variant, the buried amino group of the Lys38 side chain makes an ion pair with Glu122, whereas in the structure of the L38E variant, the buried carboxyl group of Glu38 interacts with two backbone amides and has several nearby carboxyl oxygen atoms. Previously, we showed that the pKa of Lys38 is normal owing to structural reorganization and water penetration concomitant with ionization of the Lys side chain. In contrast, the pKa values of Asp38 and Glu38 are perturbed significantly owing to an imbalance between favorable polar interactions and unfavorable contributions from dehydration and from Coulomb interactions with surface carboxylic groups. Their ionization is also coupled to subtle structural reorganization. These results illustrate the complex interplay between local polarity, Coulomb interactions, and structural reorganization as determinants of pKa values of internal groups in proteins. This study suggests that improvements to computational methods for pKa calculations will require explicit treatment of the conformational reorganization that can occur when internal groups ionize. 相似文献
14.
The effect of metal ions on human activated Factor X (Factor Xa) hydrolysis of the chromogenic substrate benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (S2222) was studied utilizing initial rate enzyme kinetics. The divalent metal ions Ca2+, Mn2+, and Mg2+ enhanced Factor Xa amidolytic activity with Km values of 30 μm, 20 μm, and 1.4 mm, respectively. Na+ activation of Factor Xa amidolytic activity was also found. The Km for Na+ activation was 0.31 m. Both the divalent metal ions and Na+ increased the affinity of Factor Xa for S2222 and had no effect on the maximal velocity of the reaction. Other monovalent cations were unable to activate Factor Xa. However, K+ was a competitive inhibitor of the Na+ activation (Ki = 0.14 m). Lanthanide ions inhibited Factor Xa amidolytic activity. Gd3+ inhibition of Factor Xa hydrolysis of S2222 was noncompetitive and had a Ki of 3 μm. The lanthanide ion inhibition could not be reversed by Ca2+ even when Ca2+ was present in a 1000-fold excess over its Km indicating nonidentity of the Factor Xa lanthanide and Ca2+ binding sites. It is concluded that the Factor Xa Ca2+ binding sites have characteristics different from those previously described for the Factor X molecule and that Mg2+, Na+, and K+ may be physiological regulators of Factor Xa activity. 相似文献
15.
J.W. Gorrod 《Chemico-biological interactions》1973,7(5):289-303
The various types of nitrogen which occur in organic compounds and which are susceptible to biological oxidation are clearly divided into groups depending upon the pKa, of the constituent nitrogen. The enzymatic processes which give rise to the N-oxidation products are reviewed by a consideration of species differences, age of animal, pH optima, influence of inducing agents, inhibitors and microsomal pretreatments, as well as the stereochemistry of the nitrogen atom.From the data collected, a concept is developed which suggests that all basic amines (group I) are oxidised by a flavine adenine nucleotide (FAD)-dependent enzyme system, whereas non-basic nitrogen-containing compounds (group III) are oxidised by a cytochrome P450-dependent system.It is further suggested that compounds of intermediary pKa,i.e. between 1 and 7 (group II), may be substrates for both enzyme systems, which would yield the same products, but by different processes. The extent to which N-oxidation occurs in a species would therefore depend on the pKa of the substrate and the amounts and ratio of the two enzymes present, a lower pKa favouring oxidation by the cytochrome P450 system and a higher pKa favouring oxidation by the FAD system.In a similar manner, it is suggested that the oxidation of aromatic heterocyclic amines depends upon the pKa of the nitrogen, compounds having a low pKa being preferentially metabolised by nitrogen oxidation. 相似文献
16.
The maximal velocity, V, for isocitrate cleavage by isocitrate lyase from Neurospora crassa is dependent on two dissociable groups with pKa values of 6.1 and 8.6. A dissociable group with a pKa of 8.5 on the enzyme-substrate complex affects the pKm for isocitrate. The pKi for homoisocitrate is affected in a like manner. The pH dependence of the pKi's for succinate, a product of isocitrate cleavage, and the succinate analog maleate is similar to the pH dependence of the pKm of isocitrate below pH 7.3, but is markedly different above this pH. Both the Km for isocitrate and the Ki for succinate were dependent upon Mg2+ concentration. The pKi for oxalate, an analog of glyoxylate which is also a product of isocitrate cleavage, is dependent on a group with a pKa of 6.8 on the enzyme-inhibitor complex. The pH dependence of the pKi for phosphoenolpyruvate, which binds to the succinate site, suggests that it is dependent on two dissociable groups, one on phosphoenolpyruvate and one, by analogy to the pKm for isocitrate, on the enzyme-glyoxylate-inhibitor complex. 相似文献
17.
The maximal velocity, V, for isocitrate cleavage by isocitrate lysase from Pseudomonas indigofera was dependent on two dissociable groups (pKa's of 6.9 and 8.6). The pH dependence of the pKi for succinate, a product of isocitrate cleavage, implied that a dissociable group (pKa of 6.0) on the enzyme functions in binding succinate. The pKi's for maleate and itaconate (succinate analogs) were similarly pH dependent. The pKi for oxalate, an analog of glyoxylate which is also a product of isocitrate cleavage, was pH independent. In contrast the pKi's of the four-carbon dicarboxylic acid inhibitors, fumarate and meso-tartrate, both of which affect the glyoxylate site, were dependent on a dissociable group on the enzyme-inhibitor complex. Comparison of the pH dependence of the pKm for isocitrate and the pKi for succinate (and succinate analogs) indicated that the binding of isocitrate was dependent on an acidic dissociable group on the enzyme (pKa of 5.8). The pH dependence of the pKi for homoisocitrate was similar. In addition the Ki for succinate and Km for isocitrate were dependent upon Mg2+ concentration. Inhibition by phosphoenolpyruvate, which binds to the succinate site and may regulate isocitrate lyase from P. indigofera, was twice as pH dependent as that for succinate. Two dissociable groups, one on the enzyme (pKa of 5.8) and one on phosphoenolpyruvate (pKa of 6.35), contributed to the pH dependence observed with phosphoenolpyruvate. 相似文献
18.
A model for a main element of the active site of skeletal muscle myosin is presented that relates directly to the 92 amino acid fragment (p10) of myosin recently described by Elzinga &; Collins (1977). In this model, the substrate, an eight-membered cyclic complex of MgATP, fits tightly into a 16 amino acid segment of p10 and interacts with seven of its amino acids. A main feature of the model is the important role played by the one molecule of Nτ-methylhistidine2 that is present in each myosin heavy chain. At the site, it is postulated that this rare amino acid functions as a donor ligand to Mg2+. Once Nτ-methylhistidine is put in place next to the metal, the other amino acids that appear to form a pocket come easily into position around the MgATP. These amino acids with their postulated functions are: tyrosine 72, which through a Mg-bound water, or perhaps directly, is attached to the Mg; histidine 76, which donates a proton to the Pγ of ATP; lysine 78, which binds electrostatically to Pβ of ATP; phenylalanines 80 and 81, which flank the purine ring of ATP; and aspartate 66, which forms a hydrogen bond to the 6-amino group of adenine. The Mg-coordination role ascribed to Nτ-methylhistidine 69 in skeletal muscle myosin could be taken by histidine 69 in cardiac myosin and in other muscle myosins that do not contain the methylated amino acid.The choice of p10 to contain a main element of the active site is based on: (a) the presence in p10 of the essential sulfhydryl groups, SH1 and SH2, whose modification affects the ATPase activity of myosin; (b) the presence in ρ10 of Nτ-methylhistidine, an unusual amino acid whose methylation in skeletal muscle we take as an indicator for a special function at the active site; (c) the position of p10 in the primary structure near the junction between subfragment 1 and subfragment 2 (the hinge region) where, we postulate, enzymatic events at the active site are coupled to movements of the hinge that occur during contraction; (d) indications that the DTNB light chain, probably involved in regulation, is also near the hinge; (e) the effects of MgATP at the active site on the chemical reactivity of three SH groups (SH1, SH2 and SH3) located near the hinge; and (f) the effect of hinge cleavage on the oxygen exchange reaction catalyzed at the active site. The correlation of all these observations forms the basis for our placement of part of the active site on p10 near the subfragment 1-subfragment 2 hinge. 相似文献
19.
Jonathan W. Day Chan Hyuk Kim Vaughn V. Smider Peter G. Schultz 《Bioorganic & medicinal chemistry letters》2013,23(9):2598-2600
The bidentate metal binding amino acid bipyridylalanine (BpyAla) was incorporated into a disulfide linked cyclic peptide phage displayed library to identify metal ion binding peptides. Selection against Ni2+–nitrilotriacetic acid (NTA) enriched for sequences containing histidine and BpyAla. BpyAla predominated when selections were carried out at lower pH, consistent with the differential pKa’s of histidine and BpyAla. Two peptides containing BpyAla were synthesized and found to bind Ni2+ with low micromolar dissociation constants. Incorporation of BpyAla and other metal binding amino acids into peptide and protein libraries should enable the evolution of novel binding and catalytic activities. 相似文献
20.
The pKa values of two histidine residues in human haemoglobin, the Bohr effect, and the dipole moments of alpha-helices 总被引:4,自引:0,他引:4
M F Perutz A M Gronenborn G M Clore J H Fogg D T Shih 《Journal of molecular biology》1985,183(3):491-498
Studies of abnormal and chemically modified haemoglobins indicate that in 0.1 m-NaCl about 40% of the alkaline Bohr effect of human haemoglobin is contributed by the C-terminal histidine HC3(146)β. In deoxyhaemoglobin, the imidazole of this histidine forms a salt bridge with aspartate FG1(94)β, in oxyhaemoglobin or carbonmonoxyhaemoglobin it accepts a hydrogen bond from its own NH group instead. Kilmartin et al. (1973) showed that in 0.2 m-NaCl + 0.2 m-phosphate this change of ligation lowered the pKa of the histidine from 8.0 in Hb3 to 7.1 in HbCO, but Russu et al. (1980) claimed that in bis-Tris buffer without added NaCl its pKa in HbCO dropped no lower than 7.85, and that in this medium the C-terminal histidine made only a negligible contribution to the alkaline Bohr effect.We have compared the histidine resonances of HbCO A with those of three abnormal haemoglobins: HbCO Cowtown (His HC3(146)β → Leu), HbCO Wood (His FG4(97)β → Leu) and HbCO Malmø (His FG4(97)β → Gln). Our results show that the resonance assigned by Russu et al. to His HC3(146)β in fact belongs to His FG4(97)β. Although in Hb the pKa of His HC3(146)β is 8.05 ± 0.05 independent of ionic strength, in HbCO its pKa drops sharply with diminishing ionic strength, so that in the buffer employed by Russu et al. it has a pKa of 6.2 and makes a contribution to the alkaline Bohr effect that is 57% larger than in the phosphate buffer employed by Kilmartin et al. (1973).In HbCO A, His FG4(97)β does not contribute to the Bohr effect, but in HbCO from which His HC3(146)β has been cleaved (HbCO des-His), His FG4(97)β is in equilibrium between two conformations with different pKa values. This equilibrium varies with ionic strength and pH, and presumably also with degree of ligation of the haem moiety.In HbCO A, His FG4(97)β has a pKa of 7.8 compared to the pKa value of about 6.6 characteristic of free histidines at the surface of proteins. This high pKa is accounted for by its interaction with the negative pole at the C terminus of helices F and FG. It corresponds to a free energy change of the same order as that observed in the interaction of histidines with carboxylate ions and confirms the strongly dipolar character of α-helices, which manifests itself even when they lie on the surface of the protein. 相似文献