Cucurbitacin delta 23-reductase from the fruit of Cucurbita maxima var. Green Hubbard. Physicochemical and fluorescence properties and enzyme-ligand interactions.

Cucurbitacin delta 23-reductase from Cucurbita maxima var. Green Hubbard fruit displays an apparent Mr of 32,000, a Stokes radius of 263 nm and a diffusion coefficient of 8.93 X 10(-7) cm2 X s-1. The enzyme appears to possess a homogeneous dimeric quaternary structure with a subunit Mr of 15,000. Two tryptophan and fourteen tyrosine residues per dimer were found. Emission spectral properties of the enzyme and fluorescence quenching by iodide indicate the tryptophan residues to be buried within the protein molecule. In the pH range 5-7, where no conformational changes were detected, protonation of a sterically related ionizable group with a pK of approx. 6.0 markedly influenced the fluorescence of the tryptophan residues. Protein fluorescence quenching was employed to determine the dissociation constants for binding of NADPH (Kd 17 microM), NADP+ (Kd 30 microM) and elaterinide (Kd 227 microM). Fluorescence energy transfer between the tryptophan residues and enzyme-bound NADPH was observed.     

Parallel increases in amount of (3H)GDP binding and thermogenin antigen in brown-adipose-tissue mitochondria of cafeteria-fed rats

In order to investigate the possible existence of a ‘masked’ (i.e. non-GDP-binding) form of thermogenin (the brown-adipose-tissue specific, 32 000 Da so-called “uncoupling” protein), rats were fed a routine pellet diet or, in addition to this, a cafeteria diet. Brown-adipose-tissue mitochondria isolated from the cafeteria-fed animals showed as expected an increased (³H)GDP binding capacity (from 0.26 to 0.41 nmol/mg protein; an increase of 57%). However, when analysed by a quantitative enzyme-linked immuno-assay system for thermogenin, the mitochondria also showed an increased content of thermogenin (from 14.9 to 20.5 μg per mg; an increase of 38%). The ratio between thermogenin and GDP binding was 61 000 and 53 000 g/mol in the two cases; these values were not significantly different and were in good agreement with suggestions that thermogenin binds 1 GDP per thermogenin dimer. It was concluded that under the conditions investigated, there was no reason to assume the existence of a masked form of thermogenin.     

Binding of regulatory ligands to rabbit muscle phosphofructokinase. A model for nucleotide binding as a function of temperature and pH.

The binding of nucleoside triphosphates to rabbit muscle phosphofructokinase has been determined in 0.05 M phosphate buffers by changes in intrinsic protein fluorescence and by direct binding measurements. These experiments have been performed over a wide range of pH, temperature, and effector concentration. Quenching of protein fluorescence is shown to measure binding of nucleotides to a site which is not the active site but rather a site responsible for inhibition of the kinetic activity. This site is relatively specific for either ATP or MgATP with free ATP binding about 10-fold more tightly than MgATP. A model to describe binding to this site as a function of pH and temperature is proposed. This model assumes that the apparent affinity for ATP is determined by protonation of two ionizable groups (per subunit) and that ATP binds exclusively to protonated enzyme forms. Several ligands which affect the apparent affinity for nucleotide binding at the inhibitory site act by shifting the apparent pK of the ionizable groups. NH4+ and citrate do not influence nucleotide binding to the inhibitory site. At pH 6.9 in 0.05 M phosphate, low concentrations of MgATP or MgGTP enhance the protein fluorescence due to binding at the active site. The fluorescence studies and direct binding studies show that there is one active site and one inhibitory site per subunit. As described elsewhere (Pettigrew, D. W., and Frieden, C. (1978) J. Biol. Chem. 253, 3623-3627), there is a third nucleotide binding site on each subunit which is specific for cAMP, AMP, and ADP.     

Apparent unmasking of [3H]GDP binding in rat brown-fat mitochondria is due to mitochondrial swelling

The presence of and biochemical background for the so-called 'unmasking' phenomenon in rat brown-fat mitochondria was investigated (i.e. the apparent increase in [3H]GDP binding to the 'uncoupling' protein thermogenin, without a concomitant increase in the amount of the protein). It was found that an unmasking could be observed both 1 h after norepinephrine injection and after 1 h cold stress, provided that the rats were preacclimated to 28 degrees C. The unmasking could be observed both when a filtration method and when a centrifugation method for determination of [3H]GDP-binding capacity were used; however, the absolute values were higher with the filtration method. Based on observations of slower cytochrome-c oxidase sedimentation during centrifugation, the possibility that the matrix volume of brown-fat mitochondria isolated from warm-acclimated animals was smaller than that of cold-stressed animals was investigated with 3H2O. The cold stress increased the matrix volume from being nearly non-existent to about 1 microliter/mg. A preswelling procedure in an ionic medium could similarly increase the matrix volume in mitochondria from warm-acclimated animals but was without significant effect in the already swollen mitochondria from cold-stressed animals or from animals adapted to a lower temperature. In mitochondria from warm-acclimated animals, the ionic preswelling procedure was fully able to increase the apparent amount of GDP binding to that observed in mitochondria from cold-stressed animals, but it was practically without effect on GDP binding in mitochondria from cold-stressed animals or from animals acclimated to a lower temperature. It is concluded that the apparent 'unmasking' phenomenon, observed when the tissue is less activated than in normal control situations, is not (as hitherto anticipated) due to a specific change in thermogenin as such, but is a reflection of a general mitochondrial phenomenon.     

Interaction of 6-mercapto-GTP with bovine brain tubulin. Equilibrium aspects

In the presence of glycerol, the thionucleotide 2-amino-6-mercapto-9-ribofuranosyl purine 5'-triphosphate (S6-GTP) promotes the assembly of 6 S tubulin to form microtubules. Microtubules assembled with this analog show normal stability properties. In the absence of glycerol, few microtubules are formed with S6-GTP; however, many twisted ribbons are evident. Binding of S6-GTP to tubulin from which the associated proteins and exchangeable nucleotide have been removed (Tu(-] produces about a 16% quenching of intrinsic tubulin fluorescence. Fluorescence titrations indicate an apparent Kd for the tubulin S6-GTP complex of about 3 X 10(-8)M. Binding of S6-GTP to Tu(-) also produces a change in its absorption spectrum. The observed difference spectrum has a maximum at 350 nm and negative extrema at 323 and 338 nm. This suggests that the environment of the thioguanine ring is relatively hydrophobic. Competitive displacement studies yield apparent Kd values of about 1.7 X 10(-8)M for GTP and 8.3 X 10(-8) M for GDP. The changes in absorbance and fluorescence which accompany binding provide an excellent approach to the study of the kinetics and mechanisms of nucleotide binding as well as studies of the kinetics of displacement of GTP, GDP, and their analogs.     

Thermodynamic linkage between the binding of protons and inhibitors to HIV-1 protease

The aspartyl dyad of free HIV-1 protease has apparent pK(a)s of approximately 3 and approximately 6, but recent NMR studies indicate that the aspartyl dyad is fixed in the doubly protonated form over a wide pH range when cyclic urea inhibitors are bound, and in the monoprotonated form when the inhibitor KNI-272 is bound. We present computations and measurements related to these changes in protonation and to the thermodynamic linkage between protonation and inhibition. The Poisson-Boltzmann model of electrostatics is used to compute the apparent pK(a)s of the aspartyl dyad in the free enzyme and in complexes with four different inhibitors. The calculations are done with two parameter sets. One assigns epsilon = 4 to the solute interior and uses a detailed model of ionization; the other uses epsilon = 20 for the solute interior and a simplified representation of ionization. For the free enzyme, both parameter sets agree well with previously measured apparent pK(a)s of approximately 3 and approximately 6. However, the calculations with an internal dielectric constant of 4 reproduce the large pKa shifts upon binding of inhibitors, but the calculations with an internal dielectric constant of 20 do not. This observation has implications for the accurate calculation of pK(a)s in complex protein environments. Because binding of a cyclic urea inhibitor shifts the pK(a)s of the aspartyl dyad, changing the pH is expected to change its apparent binding affinity. However, we find experimentally that the affinity is independent of pH from 5.5 to 7.0. Possible explanations for this discrepancy are discussed.     

Specificity of nucleotide binding sites in isolated chloroplast coupling factor (CF1).

The binding of various nucleotides to chloroplast coupling factor CF1 was studied by two dialysis techniques. It was found that the number of nucleoside diphosphate sites and their specificities for the base moiety is dependent on the magnesium concentration. In the presence of 50 micrometer added MgCl2, the protein has a single strong site/mol protein with Kd = 0.5 micrometer for ADP and high specificity (Kd greater than 20 micrometer for epsilonADP, GDP, CDP). In the presence of 5 mM MgCl2, the protein has two independent tight ADP sites (Kd = 0.4 micrometer) of low specificity (Kd approximately 0.8, 2, and 2 micrometer, respectively for episilonADP, GDP, and CDP). These results are compared with the specificity of the partial reactions for photophosphorylation.     

Human plasma gelsolin reversibly binds Mg-ATP in Ca(2+)-sensitive manner.

Gelsolin is a Ca(2+)-regulated actin-modulating protein found in a variety of cellular cytoplasm and also in blood plasma. Affinity separation of human plasma gelsolin was successfully accomplished by eluting the protein with a low concentration of nucleoside polyphosphate from immobilized Cibacron Blue F3GA (1, 2). This finding was followed by the demonstration that the protein had one class of ATP binding site with Kd = 2.8 x 10(-7) M, which saturated at an ATP/gelsolin ratio of 0.6 in the absence of Ca2+ (3). To obtain further information on the nucleotide binding properties of gelsolin, binding studies were done in the presence of EGTA with GTP, ADP, and GDP by equilibrium dialysis. Incubation of plasma gelsolin with GTP resulted in binding of 0.6 mol of GTP per mol of protein with a dissociation constant of 1.8 x 10(-6) M, indicating that ATP binds to gelsolin with higher affinity than GTP. Neither ADP nor GDP at up to 100 microM appreciably bound to gelsolin at a physiological salt concentration. Then, the effects of divalent metal ions on the ATP binding to plasma gelsolin were examined. Gelsolin bound to ATP with Kd = 2.4 x 10(-6) M in a solution containing 2 mM MgCl2, whereas micromolar free Ca2+ concentrations inhibited ATP binding. Furthermore, addition of Ca2+ rapidly reversed the preformed nucleotide binding to gelsolin, suggesting that Ca2+ binding to gelsolin leads to a conformational change which disrupts a nucleotide binding fold in the protein molecule.     

Interaction of nucleotides and cations with the (Ca2+, Mg2+)-ATPase of sarcoplasmic reticulum as determined by fluorescence changes of bound 1-anilino-8-naphthalenesulfonate

The changes in fluorescence of 1-anilino-8-naphthalenesulfonate (ANS-) have been used to determine binding of ligands to the (Ca2+, Mg2+)-ATPase of sarcoplasmic reticulum vesicles, isolated from rabbit skeletal muscle. ANS- binds to sarcoplasmic reticulum membranes with an apparent Kd of 3.8 X 10(-5) M. The binding of ANS- had no effect on Ca2+ transport or Ca2+-dependent ATPase activity. EGTA, by binding endogenous Ca2+, increased the fluorescence intensity of bound ANS- by 10-12%. Subsequent addition of ATP, ADP, or Ca2+, in the presence or absence of Mg2+, reversed this change of fluorescence. The binding parameters, as determined by these decreases in fluorescence intensity, were as follows: for ATP, Kd = 1.0 X 10(-5) M, nH = 0.80; for ADP, Kd = 1.2 X 10(-5) M, nH = 0.89; and for Ca2+, Kd = 3.4 X 10(-7) M, nH = 1.8. The binding parameters for ITP and for the nonhydrolyzable analogue, adenyl-5'-yl-beta, gamma-methylene)diphosphate, were similar to those of ATP, but GDP, IDP, CDP, AMP, and cAMP had lower apparent affinities. Millimolar concentrations of pyrophosphate also decreased the fluorescence of bound ANS-, whereas orthophosphate caused a small (2-3%) increase in fluorescence in Ca2+-free media. Vanadate, in the presence of EGTA, decreased the fluorescence of bound ANS-with half-maximal effect at 4 X 10(-5) M. The changes of fluorescence intensity of bound ANS- appear to reflect conformational changes of the (Ca2+, Mg2+)-ATPase, consequent to ligand binding, with the low and high fluorescence intensity species corresponding to the E1 and E2 conformations, respectively. These appear to reflect similar conformational states of the (Ca2+, Mg2+)-ATPase to those reported by changes in intrinsic tryptophan fluorescence (DuPont, Y. (1976) Biochem, Biophys. Res. Commun. 71, 544-550).     

The interaction of human glycophorin with 8-anilino-1-naphthalene sulfonate.

Both the sialoglycoprotein of human erythrocyte membranes, glycophorin, and the sialic acid free protein, obtained by treatment of glycophorin with neuraminidase (EC 3.2.1.18), increase the fluorescence of 8-anilino-1-naphthalene sulfonate (ANS). Binding of ANS to glycophorin is weak compared with the binding to bovine serum albumin (BSA). equilibrium dialysis gives an apparent binding constant of about 4 X 10(3) M(-1) at neutral pH, but Ka increases 1.75 times when NaCl or CaCl2 are added and 10-fold when the pH is lowered to 3.0. Sialic acid groups do not significantly affect ANS binding, although they have some effect at low ionic strength and neutral pH. Fluorescence studies indicate only one to two binding sites for ANS, with apparent pK = 3.8 +/- 0.2, and located close to aromatic residues in glycophorin. Polarization and quantum efficiency of the fluorescence of ANS associated with glycophorin fail to indicate changes in the vicinity of the binding site when the pH is lowered.     

Mechanism of binding of horse liver alcohol dehydrogenase and nicotinamide adenine dinucleotide

The binding of NAD+ to liver alcohol dehydrogenase was studied by stopped-flow techniques in the pH range from 6.1 to 10.9 at 25 degrees C. Varying the concentrations of NAD+ and a substrate analogue used to trap the enzyme-NAD+ complex gave saturation kinetics. The same maximum rate constants were obtained with or without the trapping agent and by following the reaction with protein fluorescence or absorbance of a ternary complex. The data fit a mechanism with diffusion-controlled association of enzyme and NAD+, followed by an isomerization with a forward rate constant of 500 s-1 at pH 8: E E-NAD+ *E-NAD+. The isomerization may be related to the conformational change determined by X-ray crystallography of free enzyme and enzyme-coenzyme complexes. Overall bimolecular rate constants for NAD+ binding show a bell-shaped pH dependence with apparent pK values at 6.9 and 9.0. Acetimidylation of epsilon-amino groups shifts the upper pK to a value of 11 or higher, suggesting that Lys-228 is responsible for the pK of 9.0. Formation of the enzyme-imidazole complex abolishes the pK value of 6.9, suggesting that a hydrogen-bonded system extending from the zinc-bound water to His-51 is responsible for this pK value. The rates of isomerization of E-NAD+ and of pyrazole binding were maximal at pH below a pK of about 8, which is attributable to the hydrogen-bonded system. Acetimidylation of lysines or displacement of zinc-water with imidazole had little effect on the rate of isomerization of the E-NAD+ complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spectroscopic studies of the interactions of coenzymes and coenzyme fragments with pig heart, oxidized triphosphopyridine nucleotide specific isocitrate dehydrogenase

Spectroscopic, ultrafiltration, and kinetic studies have been used to characterize interactions of reduced and oxidized triphosphopyridine nucleotides (TPNH and TPN), 2'-phosphoadenosine 5'-diphosphoribose (Rib-P2-Ado-P), and adenosine 2',5'-bisphosphate [Ado(2',5')P2] with with TPN-specific isocitrate dehydrogenase. Close similarity of the UV difference spectra and of the protein fluorescence changes accompanying the formation of the binary complexes provides evidence for the binding of these nucleotides to the same site on the enzyme. From the pH dependence of the dissociation constants for TPNH binding to TPN-specific isocitrate dehydrogenase in the absence and in the presence of Mn2+, over the pH range 5.8-7.6, it has been demonstrated that the nucleotide binds to the enzyme in its unprotonated, metal-free form. The involvement of positively charged residues, protonated over the pH range studied, has been postulated. One TPNH binding site per enzyme subunit has been measured by fluorescence and difference absorption titrations. A dramatic effect of ionic strength on binding has been demonstrated: about a 1000-fold decrease in the dissociation constant for TPNH has been observed at pH 7.6 upon decreasing ionic strength from 0.336 (Kd = 1.2 +/- 0.2 microM) to 0.036 M (Kd = 0.4 +/- 0.1 nM) in the presence and in the absence of 100 mM Na2SO4, respectively. Weak competition of sulfate ions for the nucleotide binding site has been observed (KI = 57 +/- 3 mM). The binding of TPN in the presence of 100 mM Na2SO4 at pH 7.6 is about 100-fold weaker (Kd = 110 +/- 22 microM) than the binding of the reduced coenzyme and is similarly affected by ionic strength. These results demonstrate the importance of electrostatic interactions in the binding of the coenzyme to TPN-specific isocitrate dehydrogenase. The large enhancement of protein fluorescence caused by binding of TPN and Rib-P2-Ado-P (delta Fmax = 50%) and of Ado(2',5')P2 (delta Fmax = 41%) has been ascribed to a local conformational change of the enzyme. An apparent stoichiometry of 0.5 nucleotide binding site per peptide chain was determined for TPN, Rib-P2-Ado-P, and Ado(2',5')P2 from fluorescence titrations, in contrast to one binding site per enzyme subunit determined from UV difference spectral titration and ultrafiltration experiments. Thus, the binding of one molecule of the nucleotide per dimeric enzyme molecule is responsible for the total increase in protein fluorescence, while binding to the second subunit does not cause further change.(ABSTRACT TRUNCATED AT 400 WORDS)     

Labeling the (Ca(2+)-Mg2+)-ATPase of sarcoplasmic reticulum with 4-(bromomethyl)-6,7-dimethoxycoumarin: detection of conformational changes.

The (Ca(2+)-Mg2+)-ATPase of sarcoplasmic reticulum was labeled with 4-(bromomethyl)-6,7-dimethoxycoumarin. It was shown that a single cysteine residue (Cys-344) was labeled on the ATPase, with a 25% reduction in steady-state ATPase activity and no reduction in the steady-state rate of hydrolysis of p-nitrophenyl phosphate. The fluorescence intensity of the labeled ATPase was sensitive to pH, consistent with an effect of protonation of a residue of pK 6.8. Fluorescence changes were observed on binding Mg2+, consistent with binding to a single site of Kd 4 mM. Comparable changes in fluorescence intensity were observed on binding ADP in the presence of Ca2+. Binding of AMP-PCP produced larger fluorescence changes, comparable to those observed on phosphorylation with ATP or acetyl phosphate. Phosphorylation with P(i) also resulted in fluorescence changes; the effect of pH on the fluorescence changes was greater than that on the level of phosphorylation measured directly using [32P]P(i). It is suggested that different conformational states of the phosphorylated ATPase are obtained at steady state in the presence of Ca2+ and ATP and at equilibrium in the presence of P(i) and absence of Ca2+.     

Effects of fasting and food restriction on brown adipose tissue composition in normal and dystrophic hamsters

Fasting for 36-48 h or food restriction (30% reduction of daily food intake for 6 weeks) caused brown adipose tissue (BAT) atrophy in hamsters. Fasting-induced atrophy was characterized by reductions in tissue mass, DNA, protein, and thermogenin. By contrast, food restriction had no effect on tissue cellularity (DNA) but markedly reduced the tissue protein and thermogenin contents. The concentration of thermogenin in isolated mitochondria was unchanged by fasting or food restriction. Dystrophic hamsters had a reduced BAT mass when compared with weight-matched control hamsters. This resulted from a reduction in tissue cellularity since BAT DNA, protein and thermogenin contents were all reduced. The extent of binding of [3H]guanosine diphosphate to isolated mitochondria and their content of thermogenin were similar in normal and dystrophic hamsters. In response to cold exposure, as in normal hamsters, BAT of dystrophic hamsters grew and the tissue thermogenin increased, but the mitochondrial concentration of thermogenin did not change. In response to fasting, in contrast with normal hamsters, there was no significant reduction in BAT DNA in dystrophic animals and the loss of tissue protein was reduced. However, the relative changes in BAT composition during chronic food restriction were similar in normal and dystrophic animals. Thus, reduction in hamster BAT thermogenic capacity during food deprivation may occur by loss of cells and (or)reduction in the tissue protein and thermogenin contents. The extent of protein and (or) DNA loss may be dependent upon the original tissue mass and the severity of food deprivation.     

pH dependence of the kinetic properties of allosteric phosphofructokinase from Escherichia coli.

The pH dependence of the activity of the allosteric phosphofructokinase from Escherichia coli has been studied in the pH range from 6 to 9, in the absence or presence of allosteric effectors. The sigmoidal cooperative saturation of phosphofructokinase by fructose 6-phosphate has been analyzed according to the Hill equation, and the following results have been obtained: (i) the apparent affinity for Fru-6P, as measured by the half-saturating concentration, [Fru-6P]0.5, does not change with pH; (ii) the cooperativity, as measured empirically by the Hill coefficient, nH, increases markedly with pH and reaches a value of 5.5-6 at pH 9; (iii) the catalytic rate constant, kcat, is controlled by the ionization of a critical group which has a pK of 7 in the absence of effector and must be deprotonated for phosphofructokinase to be active. The observation that pH affects both the cooperativity and the maximum velocity suggests that the catalytic efficiency of a given active site could be modified by the binding of fructose 6-phosphate to other remote sites. Finding values of the cooperativity coefficient larger than the number of substrate binding sites indicates that slow conformational changes may occur in phosphofructokinase. The cooperative saturation of phosphofructokinase by fructose 6-phosphate appears more complex than described by the classical concerted model at steady state and could involve two slowly interconverting states which differ in both their turnover rate constants and their affinities for fructose 6-phosphate. The presence of GDP shifts the pK of the critical group which controls kcat from 7 to 6.6.(ABSTRACT TRUNCATED AT 250 WORDS)     

B lymphocytes from hyporesponsive SJL mice contain aberrant nucleoside binding sites

C8-substituted guanine ribonucleosides activate B cells by a novel pathway that apparently is independent of GTP-binding proteins and protein kinase C. B lymphocytes from SJL mice are hyporesponsive to antigen-independent inductive signals transmitted by these nucleosides. In the current studies, the basis for this observation was explored. Responses of normal murine strains to these agents have been dissociated into antigen-independent (inductive) and antigen-dependent (differentiative) types by use of the 7,8-disubstituted guanine ribonucleosides. Dose-response profiles for inductive responses appear to correlate with apparent Kd values for low-affinity nucleoside binding sites; dose-response curves for antigen-dependent differentiative responses correlate with apparent Kd values for high-affinity binding sites. It was found that the SJL low-affinity site exhibits an apparent Kd that is approximately 10- to 20-fold lower in affinity for 8BrGuo than that of normal CBA mice. Although the low-affinity site in normal murine strains displays nearly equivalent affinity toward C8-substituted and 7,8-disubstituted nucleosides, the low-affinity site of SJL mice binds 7,8-disubstituted compounds with approximately 5-fold higher affinity than it does monosubstituted compounds. The dissociation constant for high-affinity nucleoside binding sites of SJL mice was only slightly different from that of CBA mice, consistent with the observation of essentially normal antigen-dependent nucleoside-mediated activity in SJL mice. The current observations support (a) a role for low-affinity binding sites in antigen-independent inductive events, (b) a role for high-affinity binding sites in antigen-dependent differentiative events mediated by substituted guanine nucleosides, and (c) the existence of aberrant low-affinity binding sites in B cells from SJL mice.     

Oxidation of phenethylamine derivatives by cytochrome P450 2D6: the issue of substrate protonation in binding and catalysis.

Cytochrome P450 (P450) 2D6 oxidizes a wide variety of drugs typically at a distance of 5-7 A from a basic nitrogen on the substrate. To investigate the determinants of P450 2D6 catalysis, we analyzed the binding and oxidation of phenethylamine substrates. P450 2D6 discriminated between the various phenethylamines, as evidenced by binding and steady-state results. Whereas the spectral binding affinity for 3-methoxyphenethylamine and 4-methoxyphenethylamine was similar, the affinity for 4-hydroxyphenethylamine was 12-fold weaker than for 3-hydroxyphenethylamine at pH 7.4. The binding of 3,4-dihydroxyphenethylamine was equally poor. These equilibrium dissociation constants were based on the observation of both type I and type II perturbation difference spectra; the former involves displacement of the proximal H(2)O ligand, yielding an iron spin state change, and the latter requires nitrogen ligation to the heme iron. One explanation for the observed type II binding spectra is the presence of both protonated and unprotonated forms of these compounds. To address this possibility, the K(S) values for 3-methoxyphenethylamine and 4-methoxyphenethylamine were determined as a function of pH. Two apparent pK(a) values were determined, which corresponded to a P450 2D6 residue involved in binding and to a lowered pK(a) of a substrate amine group upon binding P450 2D6. The apparent pK(a) of the enzyme residue (6.6) is much higher than the expected pK(a) of Asp301, which has been hypothesized to play a role in binding. Interestingly, the apparent pK(a) for the methoxyphenethylamine derivatives decreased by as much as 2 pH units upon binding to P450 2D6. 3-Methoxyphenethylamine and 4-methoxyphenethylamine underwent sequential oxidations with O-demethylation and subsequent ring hydroxylation to form 3,4-dihydroxyphenethylamine (dopamine). At higher substrate concentrations, the second oxidation was inhibited. This result can be explained by the increasing concentration of the inhibitory unprotonated substrate. Nevertheless, the rates of methoxyphenethylamine oxidations are the highest reported for P450 2D6 substrates.     

Histidine pK(a) values for the N-lobe of human transferrin: effect of substitution of binding site Asp by Ser (D63S)

The pK(a) values have been determined for eight of the nine histidine residues and the amino terminus of the N-lobe of human apo-transferrin (hTF/2N), and for seven of the nine histidine residues and the amino terminus of the protein Asp63Ser hTF/2N containing a mutation of the Fe(3+)-ligand Asp63 to Ser63. Calculations suggested that substitution of aspartate by serine would result in decreases of the pK(a) values of most of the histidine residues in the protein. This was found to be the case experimentally, and allowed assignment of the varepsilonCH resonance of His249. For the wild-type protein, the His residue with a pK(a) of 7.40 was assigned as His249, whereas for the mutant, no observable His residue had a pK(a) value higher than 6.9. The protonated form of His249 appears to be stabilised by interactions with Asp63, and the high pK(a) value may be critical for ensuring the release of iron at endosomal pH (5.5). The mutation lowered the apparent binding constant of hTF/2N for the synergistic anion oxalate from log K 4.0 to log K 3.3. (1)H NMR spectral changes induced by Ga(3+) binding to the mutant are compared to those observed for the wild-type protein.     

Ionization behavior of acidic residues in calbindin D(9k).

The ionization state of seven glutamate residues, one aspartate, and the C-terminal alpha-COOH group in bovine apo calbindin D(9k) has been studied by measurement and modeling of the pH titration curves and apparent pK(a) values. The observed pK(a) ranged from 3.0 to 6.5. Most of the observed acidic groups were half-ionized at lower pH values than those in unstructured proteins. As a rule, the ionization equilibria extended over a wider pH range than in the case of unperturbed single titrations, indicating a complex influence of protein charges on the charge state of each individual residue. Glu17, which is a backbone Ca(2+)-ligand in the N-terminal binding loop of calbindin D(9k), was half-protonated at pH 3.6 but manifested biphasic titration with apparent pK(a) values of 3.2 and 6.5. Complementary Monte Carlo simulations of the titration process and pK(a) values of the acidic groups in calbindin D(9k) reproduce the experimentally observed titration features, except for the pronounced double titration of Glu17. Discrepancies between the results from direct measurement and from modeling may be partly caused by changes in the protein structure when the net charge changes from -8 to +11 over the isoelectric point at pH 5. Proteins 1999;37:106-115.