Effects of external pH on substrate binding and on the inward chloride translocation rate constant of band 3

To test the hypothesis that amino acid residues in band 3 with titratable positive charges play a role in the binding of anions to the outside-facing transport site, we measured the effects of changing external pH (pH(O)) on the dissociation constant for binding of external iodide to the transport site, K(O)(I). K(O)(I) increased with increasing pH(O), and a significant increase was seen even at pH(O) values as low as 9.9. The dependence of K(O)(I) on pH(O) can be explained by a model with one titratable site with pK 9.5 +/- 0.2 (probably lysine), which increases anion affinity for the external transport site when it is in the positively charged form. A more complex model, analogous to one recently proposed by Bjerrum (1992), with two titratable sites, one with pK 9.3 +/- 0.3 (probably lysine) and another with pK > 11 (probably arginine), gives a slightly better fit to the data. Thus, titratable positively charged residues seem to be functionally important for the binding of substrate anions to the outward-facing anion transport site. In addition, analysis of Dixon plot slopes for L inhibition of Cl- exchange at different pH 0 values, coupled with the assumption that pH(O) has parallel effects on external I- and Cl- binding, indicates that k', the rate-constant for inward translocation of the complex of Cl- with the extracellular transport site, decreases with increasing pH(O). The data are compatible with a model in which titration of the pK 9.3 residue decreases k to 14 +/- 10% of its value at neutral pH(O). This result, however, together with Bjerrum's (1992) observation that the maximum flux J(M)) increases 1.6- fold when this residue is deprotonated, makes quantitative predictions that raise significant questions about the adequacy of the two titratable site ping-pong model or the assumptions used in analyzing the data.     

Dopa decarboxylase exhibits low pH half-transaminase and high pH oxidative deaminase activities toward serotonin (5-hydroxytryptamine)

Dopa decarboxylase (DDC) catalyzes not only the decarboxylation of L-aromatic amino acids but also side reactions including half-transamination of D-aromatic amino acids and oxidative deamination of aromatic amines. The latter reaction produces, in equivalent amounts, an aromatic aldehyde or ketone (depending on the nature of the substrate), and ammonia, accompanied by O(2) consumption in a 1 : 2 molar ratio with respect to the products. The kinetic mechanism and the pH dependence of the kinetic parameters have been determined in order to obtain information on the chemical mechanism for this reaction toward 5-hydroxytryptamine (5-HT). The initial velocity studies indicate that 5-HT and O(2) bind to the enzyme sequentially, and that D-Dopa is a competitive inhibitor versus 5-HT and a noncompetitive inhibitor versus O(2). The results are consistent with a mechanism in which 5-HT binds to DDC before O(2). The pH dependency of log V for the oxidative deaminase reaction shows that the enzyme possesses a single ionizing group with a pK value of approximately 7.8 that must be unprotonated for catalysis. In addition to an ionizing residue with a pK value of 7.9 similar to that found in the V profile, the (V/K)(5-HT) profile exhibits a pK value of 9.8, identical to that of free substrate. This pK was therefore tentatively assigned to the alpha-amino group of 5-HT. No titratable ionizing residue was detected in the (V/K)(O2) profile, in the pH range examined. Surprisingly, at pH values lower than 7, where oxidative deamination does not occur to a significant extent, a half-transamination of 5-HT takes place. The rate constant of pyridoxamine 5'-phosphate formation increases below a single pK of approximately 6.7. This value mirrors the spectrophotometric pK(spec) of the shift 420-384 nm of the external aldimine between DDC and 5-HT. Nevertheless, the analysis of the reaction of DDC with 5-HT under anaerobic conditions indicates that only half-transamination occurs with a pH-independent rate constant over the pH range 6-8.5. A model accounting for these data is proposed that provides alternative pathways leading to oxidative deamination or half-transamination.     

Modulation of serotonin uptake kinetics by ions and ion gradients in human placental brush-border membrane vesicles

The modulation of serotonin uptake kinetics by Na+, Cl-, H+, and K+ was investigated in brush-border membrane vesicles prepared from normal human term placentas. The presence of Na+ and Cl- in the external medium was mandatory for the function of the serotonin transporter. In both cases, the initial uptake rate of serotonin was a hyperbolic function of the ion concentration, indicating involvement of one Na+ and one Cl- per transport of one serotonin molecule. The apparent dissociation constant for Na+ and Cl- was 145 and 79 mM, respectively. The external Na+ increased the Vmax of the transporter and also increased the affinity of the transporter for serotonin. The external Cl- also showed similar effects on the Vmax and the Kt, but its effect on the Kt was small compared to that of Na+. The presence of an inside-acidic pH, with or without a transmembrane pH gradient, stimulated the NaCl-dependent serotonin uptake. The effect of internal [H+] on the transport function was to increase the Vmax and decrease the affinity of the transporter for serotonin. The presence of K+ inside the vesicles also greatly stimulated the initial rates of serotonin uptake, and the stimulation was greater at pH 7.5 than at pH 6.5. This stimulation was a hyperbolic function of the internal K+ concentration at both pH values, indicating involvement of one K+ per transport of one serotonin molecule. The apparent dissociation constant for K+ was 5.6 mM at pH 6.5 and 4.0 mM at pH 7.5. The effects of internal [K+] on the uptake kinetics were similar to those of internal [H+].(ABSTRACT TRUNCATED AT 250 WORDS)     

35Cl nuclear magnetic resonance line broadening shows that eosin-5-maleimide does not block the external anion access channel of band 3.

It has been suggested that Lys-430 of band 3, with which eosin-5-maleimide (EM) reacts, is located in the external channel through which anions gain access to the external transport site, and that EM inhibits anion exchange by blocking this channel. To test this, we have used 35Cl nuclear magnetic resonance (NMR) to measure Cl- binding to the external transport site in control and EM-treated human red blood cells. Intact cells were used rather than ghosts, because in this case all line broadening (LB) results from binding to external sites. In an NMR spectrometer with a 9.4-T magnetic field, red blood cells at 50% concentration (v/v) in 150 mM Cl- medium at 3 degrees C caused 19.0 +/- 1.2 Hz LB. Of this, 7.9 +/- 0.7 Hz was due to Cl- binding to the high affinity band 3 transport sites, because it was prevented by an apparently competitive inhibitor of anion exchange, 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS). The LB was not due to hemoglobin released from the cells, as little LB remained in the supernatant after cells were removed by centrifugation. Saturable Cl- binding remained in EM-treated cells, although the binding was no longer DNDS-sensitive, because EM prevents binding of DNDS. The lower limit for the rate at which Cl- goes from the binding site to the external medium is 2.15 x 10(5) s-1 for control cells and 1.10 x 10(5) s-1 for EM-treated cells, far higher than the Cl- translocation rate at 3 degrees C (about 400 s-1). Thus, EM does not inhibit Cl- exchange by blocking the external access channel. EM may therefore be useful for fixing band 3 in one conformation for studies of Cl- binding to the external transport site.     

The kinetics of the reactions of Parasponia andersonii hemoglobin with oxygen, carbon monoxide, and nitric oxide

Hemoglobin I was isolated from nodules formed on the roots of Parasponia andersonii inoculated with Rhizobium strain CP 283. The rate of oxygen dissociation from Parasponia hemoglobin increases about 12-fold between pH 4 and 7, with apparent pK 6.4, to reach a limiting value of 14.8s-1. The optical spectrum of oxyhemoglobin in the visible region is also dependent on pH with pK near 6.4. The rate constant for oxygen combination with Parasponia hemoglobin increases about 7-8-fold between pH 4 and 7, with apparent pK 5.37, to reach a value of 1.67 X 10(8) M-1 s-1 at pH 7. The optical spectrum of deoxyhemoglobin in the visible region and the rate constant for carbon monoxide combination are also dependent on pH with apparent pK 5.65 and 5.75, respectively. The rate constant for carbon monoxide dissociation is independent of pH. The oxygen affinity of Parasponia hemoglobin, P50 = 0.049 torr at 20 degrees C, calculated from the kinetic constants at pH 7, is very great. At alkaline pH there is a prominent geminate reaction with oxygen and nitric oxide, with both subnanosecond and tens of nanosecond components. These reactions disappear at acid pH, with pK 6.4, and the effective quantum yield is reduced. In general, the reactions of Parasponia hemoglobin with oxygen and carbon monoxide resemble those of soybean leghemoglobin. In each, great oxygen affinity is achieved by unusually rapid oxygen combination together with a moderate rate of oxygen dissociation. We suggest that protonation of a heme-linked group with pK near 6.4 controls many properties of Parasponia oxyhemoglobin, and protonation of a group with pK near 5.5 controls many properties of Parasponia deoxyhemoglobin.     

On the protein residues that control the yield and kinetics of O(630) in the photocycle of bacteriorhodopsin

The effects of pH on the yield (phi(r)), and on the apparent rise and decay constants (k(r), k(d)), of the O(630) intermediate are important features of the bacteriorhodopsin (bR) photocycle. The effects are associated with three titration-like transitions: 1) A drop in k(r), k(d), and phi(r) at high pH [pK(a)(1) approximately 8]; 2) A rise in phi(r) at low pH [pK(a)(2) approximately 4.5]; and 3) A drop in k(r) and k(d) at low pH [pK(a)(3) approximately 4. 5]. (pK(a) values are for native bR in 100 mM NaCl). Clarification of these effects is approached by studying the pH dependence of phi(r), k(r), and k(d) in native and acetylated bR, and in its D96N and R82Q mutants. The D96N experiments were carried out in the presence of small amounts of the weak acids, azide, nitrite, and thiocyanate. Analysis of the mutant's data leads to the identification of the protein residue (R(1)) whose state of protonation controls the magnitude of phi(r), k(r), and k(d) at high pH, as Asp-96. Acetylation of bR modifies the Lys-129 residue, which is known to affect the pK(a) of the group (XH), which releases the proton to the membrane exterior during the photocycle. The effects of acetylation on the O(630) parameters reveal that the low-pH titrations should be ascribed to two additional protein residues R(2) and R(3). R(2) affects the rise of phi(r) at low pH, whereas the state of protonation of R(3) affects both k(r) and k(d). Our data confirm a previous suggestion that R(3) should be identified as the proton release moiety (XH). A clear identification of R(2), including its possible identity with R(3), remains open.     

Peroxidative oxidation of halides catalysed by myeloperoxidase. Effect of fluoride on halide oxidation

It was found that all halides can compete with cyanide for binding with myeloperoxidase. The lower is the pH, the higher is the affinity of halides. The apparent dissociation constants (Kd) of myeloperoxidase-cyanide complex were determined in the presence of F-, Cl-, Br- and I- in the pH range of 4 to 7. In slightly acidic pH (4 - 6) fluoride and chloride exhibit a higher affinity towards the enzyme than bromide and iodide. Taking into account competition between cyanide and halides for binding with myeloperoxidase the dissociation constants of halide-myeloperoxidase complexes were calculated. All halides except fluoride can be oxidized by H2O2 in the presence of myeloperoxidase. However, since fluoride can bind with myeloperoxidase, it can competitively inhibit the oxidation of other halides. Fluoride was a competitive inhibitor with respect to other halides as well as to H2O2. Inhibition constants (Ki) for fluoride as a competitive inhibitor with respect to H2O2 increased from iodide oxidation through bromide to chloride oxidation.     

pH dependence of progesterone interaction with progesterone-binding globulin. Kinetic and equilibrium studies.

The kinetics of binding and dissociation for the progesterone-binding globulin (PBG)-progesterone complex have been measured as a function of pH. The association rate constant appears to be independent of pH from pH to 10 with an average value of kon = 8.5 X 10(7)M-1 S-1. The dissociation rate constant is strongly pH dependent with the dependency defined by: koff = k0 (1 + [H+]/K1 + K2/[H+])(1 + K3*/[H+])/(1 + K3/[H+]). The best values for the various parameters were k0 = 0.0785 s-1, pK1 = 5.30, pK2 = 10.54, pK3* = 7.41, and pK3 = 7.21. Simpler expressions were inadequate to fit the data, and it was concluded that at least three ionizing residues are responsible for the stability of the PBG-progesterone complex. The affinity constant was determined by equilibrium dialysis over the range of pH 3 to 12. The ratio of the association and dissociation rate constants is in agreement with the affinity constant from pH 6.5 to 10.5. The influence of pH on the conformation and binding activity of PBG was also investigated. Denaturation by acid, base, or guanidine hydrochloride leads to a reversible loss of binding activity. Regain of binding activity in all cases is slow with half-times of 0.5 to 2.7 h, depending on conditions. The rate of acid denaturation was found to be incompletely protonated at pH 1.4, suggesting a buried carboxylic acid residue. The slow renaturation of PBG might be due to the difficulty of burying a charged residue in the protein's interior coupled with steric hindrance by the large carbohydrate moiety of PBG.     

Modulation of cystic fibrosis transmembrane conductance regulator (CFTR) activity and genistein binding by cytosolic pH

Potentiators are molecules that increase the activity of the cystic fibrosis transmembrane conductance regulator (CFTR). Some potentiators can also inhibit CFTR at higher concentrations. The activating binding site is thought to be located at the interface of the dimer formed by the two nucleotide-binding domains. We have hypothesized that if binding of potentiators involves titratable residues forming salt bridges, then modifications of cytosolic pH (pH(i)) would alter the binding affinity. Here, we analyzed the effect of pH(i) on CFTR activation and on the binding of genistein, a well known CFTR potentiator. We found that pH(i) does modify CFTR maximum current (I(m)) and half-activation concentration (K(d)): I(m) = 127.7, 185.5, and 231.8 μA/cm(2) and K(d) = 32.7, 56.6 and 71.9 μm at pH 6, 7.35, and 8, respectively. We also found that the genistein apparent dissociation constant for activation (K(a)) increased at alkaline pH(i), near cysteine pK (K(a) = 1.83, 1.81 and 4.99 μm at pH(i) 6, 7.35, and 8, respectively), suggesting the involvement of cysteines in the binding site. Mutations of cysteine residues predicted to be within (Cys-491) or outside (Cys-1344) the potentiator-binding site showed that Cys-491 is responsible for the sensitivity of potentiator binding to alkaline pH(i). Effects of pH(i) on inhibition by high genistein doses were also analyzed. Our results extend previous data about multiple effects of pH(i) on CFTR activity and demonstrate that binding of potentiators involves salt bridge formation with amino acids of nucleotide-binding domain 1.     

pH-controlled hydrogen-bonding (Short Communication)

The pH-dependence of the degree of hydrogen-bonding between a base and its conjugate acid is considered. When only a small proportion of the total base is complexed, the amount complexed is proportional to (1+coshp)(-1) where p=2.303 (pK(a)-pH), pK(a) being the dissociation constant of the conjugate acid. This represents sharp pH-dependence. As the proportion complexed increases, the curve broadens, eventually becoming flat-topped, with more than half the base complexed over the range of pH values pK(a)+/-logKC, approximately. (K is the complex association constant and C is the formal base concentration, including all forms.) There are similarities to the extent of mono-protonation of a dibasic acid.     

Characteristics of the chloride conductance in muscle fibers of the rat diaphragm

In muscle fibers from the rat diaphragm, 85% of the resting membrane ion conductance is attributable to Cl-. At 37 degree C and pH 7.0, GCl averages 2.11 mmho/cm2 while residual conductance largely due to K+ averages 0.34 mmho/cm2. The resting GCl exhibits a biphasic temperature dependence with a Q10 of 1.6 between 6 degree C and 25 degree C and a Q10 of nearly 1 between 25 degree C and 40 degree C. Decreasing external pH reversibly reduced GCl; the apparent pK for groups mediating this decrease is 5.5. Increasing pH up to 10.0 had no effect on GCl. Anion conductance sequence and permeability sequence were both determined to be Cl-greater than Br-greater than or equal to I-greater than CH3SO4-. Lowering the pH below 5.5 reduced the magnitude of the measured conductance to all anions but did not alter the conductance sequence. The permeability sequence was likewise unchanged at low pH. Experiments with varying molar ratios of Cl- and I- indicated a marked interaction between these ions in their transmembrane movement. Similar but less striking interaction was seen between Cl- and Br-. Current-voltage relationships for GCl measured at early time-points in the presence of Rb+ were linear, but showed marked rectification with longer hyperpolarizing pulses (greater than 50ms) due to a slow time-and voltage-dependent change in membrane conductance to Cl-. This nonlinear behavior appeared to depend on the concentration of Cl- present but cannot be attributed to tubular ion accumulation. Tubular disruption with glycerol lowers apparent GCl but not GK, suggesting that the transverse tubule (T-tubule) system is permeable to Cl- in this species. Quantitative estimates indicate that up to 80% of GCl may be associated with the T tubules.     

Varying apparent rate constant: determination of uptake and release of protons during tetramer-dimer dissociation in human hemoglobin A

The effect of association-dissociation on the sulphydryl reactivity of human hemoglobin A is reported. The reactivity of CysF9(93)beta towards the sulphydryl reagent, 5,5'-dithiobis(2-nitrobenzoate), is higher at lower concentrations of hemoglobin at all pH values. This is because hemoglobin dimers have higher sulphydryl reactivity than tetramers and it is known that the proportion of dimers increases as the hemoglobin concentration decreases. This study takes advantage of this observation to determine the tetramer-dimer dissociation constant, K(4,2), of hemoglobin A and subsequently the proton uptake and the proton release during this process. The concentration dependence profiles of the apparent second-order rate constants, k(app), show that (between 2 and 20 microM heme) k(app) decreases with increasing hemoglobin concentration. Above 30 M heme k(app) remains fairly constant for all hemoglobin derivatives (oxy, carbonmonoxy and aquomethemoglobin) used. The pH dependence of the negative logarithm of tetramer-dimer dissociation constant, pK(4,2), for oxy- (and for carbonmonoxy-) hemoglobin exhibits a biphasic character with a maximum near pH 7.4 (and 6.6). For aquomethemoglobin, pK(4,20 decreases with increasing pH. The tetramer-dimer dissociation of human oxyhemoglobin A at an ionic strength of 200 mM uptakes 0.87 +/- 0.09 mole of protons between pH 6.2 to 7.4 phase and releases 0.84 0.09 mole of protons between pH 7.4 and 9.0 phase. Under a similar condition carbonmonoxyhemoglobin uptakes 0.54 +/- 0.05 mole of protons between pH 5.8 and 6.6 phase and releases 0.48 +/- 0.05 mole of protons between pH 6.6 and 9.0 phase. Aquomethemoglobin has only a single phase, it releases 0.39 +/- 0.05 mole of protons during tetramer-dimer dissociation.     

pH Dependence of heme iron coordination, hydrogen peroxide reactivity, and cyanide binding in cytochrome c peroxidase(H52K)

Replacement of the distal histidine, His-52, in cytochrome c peroxidase (CcP) with a lysine residue produces a mutant cytochrome c peroxidase, CcP(H52K), with spectral and kinetic properties significantly altered compared to those of the wild-type enzyme. Three spectroscopically distinct forms of the enzyme are observed between pH 4.0 and 8.0 with two additional forms, thought to be partially denatured forms, making contributions to the observed spectra at the pH extremes. CcP(H52K) exists in at least three, slowly interconverting conformational states over most of the pH range that was investigated. The side chain epsilon-amino group of Lys-52 has an apparent pK(a) of 6.4 +/- 0.2, and the protonation state of Lys-52 affects the spectral properties of the enzyme and the reactions with both hydrogen peroxide and HCN. In its unprotonated form, Lys-52 acts as a base catalyst facilitating the reactions of both hydrogen peroxide and HCN with CcP(H52K). The major form of CcP(H52K) reacts with hydrogen peroxide with a rate approximately 50 times slower than that of wild-type CcP but reacts with HCN approximately 3 times faster than does the wild-type enzyme. The major form of the mutant enzyme has a higher affinity for HCN than does native CcP.     

Effect of pH on the interaction of benzoate and D-amino acid oxidase.

The kinetic and equilibrium dissociation constants of the reversible binding of benzoate to hog kidney D-amino acid oxidase (DAAO) were studied at 19 degrees C over the pH range 5.3-10.5 by means of a stopped-flow apparatus and spectrophotometric titrations. A simple bimolecular reaction of the form second order-first order was observed; a two-step reaction was seen. Analysis of the pH dependence of the bimolecular rate constants and equilibrium dissociation constants is consistent with three ionizable groups which are important for benzoate binding. The pK values of the enzyme-related ionization are 6.3, 9.2, and 9.6. Analysis of the change in extinction coefficient at 360 nm indicates the pK of 9.6 can be assigned to the 3-imino group of the enzyme-bound flavin. The effect of benzoate on the apparent pK for the ionization of the 3-imino group of the enzyme-bound Fad has been reexamined. The presence of benzoate causes an apparent shift of this ionization from a pK value of 9.6 to 10.7.     

Interaction of t-Butylbicyclophosphorothionate with γ-Aminobutyric Acid-Gated Chloride Channels in Cultured Cerebral Neurons

The role of t-butylbicyclophosphorothionate (TBPS) as an antagonist of gamma-aminobutyric acid (GABA) was studied with primary cultures of neurons from the chick embryo cerebrum. The addition of GABA stimulated the uptake of 36Cl- by neurons and the dose dependence of this effect followed hyperbolic kinetics with a K0.5 = 1.3 microM for GABA. TBPS proved to be a potent inhibitor of GABA-dependent Cl- uptake (IC50 = 0.30 microM). Analysis of the kinetics of this process revealed that TBPS is a noncompetitive inhibitor (Ki = 0.15 microM) with respect to GABA. Scatchard analysis of direct binding of [35S]TBPS to membranes isolated from neuronal cultures gave curvilinear plots. These could be resolved by nonlinear regression methods into two components with KD values of 3.1 nM and 270 nM. The TBPS binding constant for this lower affinity site agreed well with the IC50 and Ki values for inhibition of Cl- flux, suggesting that this site is physiologically relevant to GABA antagonism. GABA was a noncompetitive displacer of [35S]TBPS binding to the lower affinity site. The Ki value for this displacement by GABA (1.7 microM) was comparable to the value for GABA enhancement of Cl- flux. The binding of [35S]TBPS to its low-affinity site on neuronal membranes was ninefold higher in the presence of Cl- than with gluconate, an impermeant anion. The rank order for anion stimulation of [35S]TBPS binding was Br- greater than or equal to SCN- greater than Cl- greater than or equal to NO3- greater than I- greater than F- greater than gluconate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of Escherichia coli ribosomal protein S8 to 16S rRNA: kinetic and thermodynamic characterization

A sensitive membrane filter assay has been used to examine the kinetic and equilibrium properties of the interactions between Escherichia coli ribosomal protein S8 and 16S rRNA. In standard conditions (0 degrees C, pH 7.5, 20 mM Mg2+, 0.35 M KCl) the apparent association constant is 5 +/- 0.5 X 10(-7) M-1. The interaction is highly specific, and the kinetics of the reaction are consistent with the apparent association constant. Nevertheless, the rate of association is somewhat slower than that expected for a diffusion-controlled reaction, suggesting some steric constraint. The association is only slightly affected by temperature (delta H = -1.8 kcal/mol). The entropy change [delta S = +29 cal/(mol K)] is clearly the main driving force for the reaction. The salt dependence of Ka reveals that five ions are released upon binding at pH 7.5 and in the presence of 10 mM magnesium. The substitution of various anions for Cl- has an appreciable effect on the magnitude of Ka, following the order CH3COO- greater than Cl- greater than Br-, thus indicating the existence of anion binding site(s) on S8. An equal number of ions were released when Cl- was replaced by CH3COO-, but the absence of anion release upon binding cannot be excluded. On the other hand, the free energy of binding appears not to be exclusively electrostatic in nature. The effect of pH on both temperature and ionic strength dependence of Ka has been examined. It appears that protonation of residue(s) (with pK congruent to 9) increases the affinity via a generalized charge effect. On the other hand, deprotonation of some residue(s) with a pK congruent to 5-6 seems to be required for binding. Furthermore, the unique cysteine present in S8 was shown to be essential for binding.     

Mechanism of nitroalkane oxidase: 2. pH and kinetic isotope effects

Nitroalkane oxidase catalyzes the oxidation of nitroalkanes to aldehydes or ketones with production of nitrite and hydrogen peroxide. pH and kinetic isotope effects with [1, 1-(2)H(2)]nitroethane have been used to study the mechanism of this enzyme. The V/K(ne) pH profile is bell-shaped. A group with a pK(a) value of about 7 must be unprotonated and one with a pK(a) value of 9.5 must be protonated for catalysis. The lower pK(a) value is seen also in the pK(is) profile for the competitive inhibitor valerate, indicating that nitroethane has no significant external commitments to catalysis. The (D)(V/K)(ne) value is pH-independent with a value of 7.5, whereas the (D)V(max) value increases from 1.4 at pH 8.2 to a limiting value of 7.4 below pH 5. The V(max) pH profile decreases at low and high pH, with pK(a) values of 6.6 and 9.5, respectively. Imidazole, which activates the enzyme, affects the V(max) but not the V/K(ne) pH profile. In the presence of imidazole at pH 7 the (D)V(max) value increases to a value close to the intrinsic value, consistent with cleavage of the carbon-hydrogen bond of the substrate being fully rate-limiting for catalysis in the presence of imidazole.     

Investigation of substrate specificity of creatine kinase using chromium (III) and cobalt(III) complexes of adenosine 5'-diphosphate

The specificity for substrate binding to creatine kinase for metal-nucleotide complexes of the type Cr-(H2O)4-n(NH3)nADP (where n = 0, 3, or 4) and Co-(H2O)4-m(NH3)mADP (for m = 3 or 4) has been investigated over the pH range 5.5-7.8 with the delta-alpha, beta-bidentate diastereoisomers. These inert nucleotide complexes acted as competitive inhibitors vs. MgADP over this range. In addition, the pH dependence of the V, V/K, and Km values for MgADP has been determined. Metal-nucleotide binding to the enzyme is strongest below an approximate pK of 6.45 but again becomes pH independent above pH 7. This pK is not associated with the metal-nucleotide complex. Instead, we conclude that the pK of the acid-base catalyst (thought to be histidine) is about 6.45 in the absence of nucleotide but is raised to 7.2 in its presence. This perturbation of the pK may result from a protein conformational change that allows a hydrogen bond to form between the phosphorylated nitrogen of phosphocreatine and the acid-base catalyst. The pK of the water in Cr(H2O)(NH3)3ADP has been determined to be 6.6, and by comparison of the binding affinity of this complex with that of Cr(NH3)4ADP or Cr(H2O)4ADP, it can be deduced that the hydroxo species binds more strongly than the aquo complex. In general, chromium nucleotides are bound more strongly than cobalt complexes, and binding affinity increases as water replaces ammonia in the first coordination sphere of the metal. Both trends are a result of stronger hydrogen-bond interactions between the metal complex and protein.     

Spectroscopic studies on the anticancer antibiotic Altromycin H and the interaction with copper(II) ions

The antitumor antibiotic Altromycin H was studied using electronic absorption (UV-Vis.) and circular dichroism (CD) spectroscopy. The dissociation constants of the phenolic groups on C(5) and C(11) were estimated as pK(1)=6.7 and pK(2)=11.8 at 25 degrees C, respectively, and a complete assignment of the CD and UV-Vis. bands is proposed. The interaction of Cu(II) ions with the Altromycin H has been also investigated by UV-Vis., CD and electron paramagnetic resonance (EPR) spectroscopy. A pH depended stepwise complex formation was observed. At pH<4 no copper-Altromycin H interactions were detected. At the 4相似文献   

Phosphorylation alters the pH-dependent active state equilibrium of rhodopsin by modulating the membrane surface potential.

Phosphorylation reduces the lifetime and activity of activated G protein-coupled receptors, yet paradoxically shifts the metarhodopsin I-II (MI-MII) equilibrium (K(eq)) of light-activated rhodopsin toward MII, the conformation that activates G protein. In this report, we show that phosphorylation increases the apparent pK for MII formation in proportion to phosphorylation stoichiometry. Decreasing ionic strength enhances this effect. Gouy-Chapman theory shows that the change in pK is quantitatively explained by the membrane surface potential, which becomes more negative with increasing phosphorylation stoichiometry and decreasing ionic strength. This lowers the membrane surface pH compared to the bulk pH, increasing K(eq) and the rate of MII formation (k(1)) while decreasing the back rate constant (k(-)(1)) of the MI-MII relaxation. MII formation has been observed to depend on bulk pH with a fractional stoichiometry of 0.6-0.7 H(+)/MII. We find that the apparent fractional H(+) dependence is an artifact of altering the membrane surface charge during a titration, resulting in a fractional change in membrane surface pH compared to bulk pH. Gouy-Chapman calculations of membrane pH at various phosphorylation levels and ionic strengths suggest MII formation behavior consistent with titration of a single H(+) binding site with 1:1 stoichiometry and an intrinsic pK of 6.3 at 0.5 degrees C. We show evidence that suggests this same site has an intrinsic pK of 5.0 prior to light activation and its protonation before activation greatly enhances the rate of MII formation.