The effects of protein hydrophobic exposure on the slope of the line in the deltapi vs pi(i) plot

The deltapi vs pi(i) curve from monolayer surface pressure detection is a powerful method to characterize the membrane insertion. The deltapi vs pi(i) curve is fixed by two parameters, the pi(c) and the slope. The intersected point (pi(c)) of deltapi vs pi(i) curve with abscissa is generally used as a quantitative measure of the membrane insertion ability of a protein. In the current work we demonstrate a correlation between the variation in the slope of the deltapi vs pi(i) curve with protein hydrophobic exposure by performing monolayer experiments on three different proteins, human apolipoprotein H, trichosanthin, and mutant trichosanthin. The value of /slope/ increases gradually following the degree of hydrophobic exposure. These findings suggest that the two parameters, the pi(c) and the slope, will complement each other to interpret the lipid/protein interaction involved in membrane insertion.     

Adsorption of bovine prothrombin to spread phospholipid monolayers

The interaction of bovine prothrombin with phospholipids was measured, using as the lipid source monolayers spread at the air-buffer interface. Fluorescence spectroscopy was implemented to determine the equilibrium concentration of free prothrombin in the aqueous subphase of the protein-monolayer suspensions, in a continuous assay system. The increase in surface pressure (pi) from the protein-monolayer adsorption was also measured and, with values of the adsorbed protein concentration (c[s]), was used to calculate dc(s)/d(pi). At a particular phosphatidylserine (PS) content of liquid-expanded (LE) phosphatidylcholine (PC)/PS monolayers, dc(s)/d(pi) was independent of the initial surface pressure (pi[i]), when this latter value exceeded 30 mN/m. However, dc(s)/d(pi) varied significantly with the relative PS content of the monolayer. Values of the equilibrium dissociation constants calculated from the concentration dependence of delta(pi) indicated that the affinity of prothrombin for LE monolayers was higher at higher PS contents and lower packing densities. The affinity of prothrombin for liquid-condensed (LC) PC/PS monolayers was found to be much weaker relative to LE monolayers of similar phospholipid composition. This approach, employing spread monolayers to study prothrombin-phospholipid binding, coupled with a simple and accurate method to determine the free protein concentration in protein-monolayer suspensions, offers significant advantages for the investigation of protein-membrane interaction. The equilibrium characteristics that describe the interaction of prothrombin with the different phospholipid monolayers under various conditions also provide support for previous results which indicated that hydrophobic interactions are involved in the adsorption of vitamin K-dependent coagulation and anticoagulation proteins to model membrane systems.     

Identification of tyrosine 79 in the tocopherol binding site of glutathione S-transferase pi

Alpha-tocopherol, the most abundant form of vitamin E present in humans, is a noncompetitive inhibitor of glutathione S-transferase pi (GST pi), but its binding site had not been located. Tocopherol iodoacetate (TIA), a reactive analogue, produces a time-dependent inactivation of GST pi to a limit of 25% residual activity. The rate constant for inactivation, k(obs), exhibits a nonlinear dependence on reagent concentration, with K(I) = 19 microM and k(max) = 0.158 min(-)(1). Complete protection against inactivation is provided by tocopherol and tocopherol acetate, whereas glutathione derivatives, electrophilic substrate analogues, buffers, or nonsubstrate hydrophobic ligands have little effect on k(obs). These results indicate that TIA reacts as an affinity label of a distinguishable tocopherol binding site. Loss of activity occurs concomitant with incorporation of about 1 mol of reagent/mol of enzyme subunit when the enzyme is maximally inactivated. Isolation of the labeled peptide from the tryptic digest shows that Tyr(79) is the only enzymic amino acid modified. The Y79F, Y79S, and Y79A mutant enzymes were generated, expressed, and purified. Changing Tyr(79) to Ser or Ala, but not Phe, renders the enzyme insensitive to inhibition by either tocopherol or tocopherol acetate as demonstrated by increases of at least 49-fold in K(I) values as compared to the wild-type enzyme. These results and examination of the crystal structure of GST pi suggest that tocopherols bind at a novel site, where an aromatic residue at position 79 is essential for binding.     

Chymotrypsin hydrolysis of X-phenyl hippurates. A quantitative structure-activity relationship and molecular graphics analysis

The hydrolysis of a set of 28 X-phenyl hippurates by chymotrypsin was investigated. From the derived Km and kcat values a quantitative structure-activity relationship was developed. This equation shows that para substituents correlated by sigma- display only an electronic effect on the formation of the ES complex whereas meta hydrophobic substituents show a hydrophobic interaction correlated by pi in addition to their electronic effect. Meta polar substituents avoid contact with the enzyme and show only electronic effects on Km. Using the x-ray crystallographic coordinates for chymotrypsin and computer graphics, a model was constructed which is used to interpret the quantitative structure-activity relationship. As with a number of previously reported examples, we have found that when polar substituents have the option of binding to hydrophobic space or remaining in the aqueous phase they follow the latter possibility.     

Separation of electronic and hydrophobic effects for the papain hydrolysis of substituted N-benzoylglycine esters

The role of hydrophobic and electronic effects on the kinetic constants kcat and Km for the papain hydrolysis of a series of 22 substituted N-benzoylglycine pyridyl esters was investigated. The series studied comprises a wide variety of substituents on the N-benzoyl ring, with about a 300,000-fold range in their hydrophobicities, and 2.1-fold range in their electronic Hammet constants (sigma). It was found that the variation in the log kcat and log 1/Km constants could be explained by the following quantitative-structure activity relationships (QSAR): log 1/Km = 0.40 pi 4 + 4.40 and log 1/kcat = 0.45 sigma + 0.18. The substituent constant, pi 4, is the hydrophobic parameter for the 4-N-benzoyl substituents. QSAR analysis of two smaller sets of glycine phenyl and methyl esters produced similar results. A clear separation of the substituent effects indicates that in the case of these particular esters, acylation appears to be the rate limiting catalytic step.     

Primary and secondary structural analyses of glutathione S-transferase pi from human placenta

The primary structure of glutathione S-transferase (GST) pi from a single human placenta was determined. The structure was established by chemical characterization of tryptic and cyanogen bromide peptides as well as automated sequence analysis of the intact enzyme. The structural analysis indicated that the protein is comprised of 209 amino acid residues and gave no evidence of post-translational modifications. The amino acid sequence differed from that of the deduced amino acid sequence determined by nucleotide sequence analysis of a cDNA clone (Kano, T., Sakai, M., and Muramatsu, M., 1987, Cancer Res. 47, 5626-5630) at position 104 which contained both valine and isoleucine whereas the deduced sequence from nucleotide sequence analysis identified only isoleucine at this position. These results demonstrated that in the one individual placenta studied at least two GST pi genes are coexpressed, probably as a result of allelomorphism. Computer assisted consensus sequence evaluation identified a hydrophobic region in GST pi (residues 155-181) that was predicted to be either a buried transmembrane helical region or a signal sequence region. The significance of this hydrophobic region was interpreted in relation to the mode of action of the enzyme especially in regard to the potential involvement of a histidine in the active site mechanism. A comparison of the chemical similarity of five known human GST complete enzyme structures, one of pi, one of mu, two of alpha, and one microsomal, gave evidence that all five enzymes have evolved by a divergent evolutionary process after gene duplication, with the microsomal enzyme representing the most divergent form.     

Hydroxyzine, promethazine and thioridazine interaction with phospholipid monomolecular layers at the air-water interface

In this work the interaction of Hydroxyzine, Promethazine and Thioridazine with Langmuir films of dipalmitoylphosphatidylcholine (dpPC) and dipalmitoylphosphatidic acid (dpPA), is studied. Temporal variations in lateral surface pressure (pi) were measured at different initial pi (pi(i)), subphase pH and drug-concentration. Drugs with the smallest (PRO) and largest (HYD) molecular size exhibited the lowest adsorption (k(a)) and the highest desorption (k(d)) rate constant values, respectively. The affinity binding constants (K(b)) obtained in monolayers followed the same profile (K(b,PRO) < K(b,HYD) < K(b,THI)) of the egg-PC/water partition coefficients (P) determined in bilayers. The drug concentration required to reach the half-maximal Deltapi at pi(i) = 14 mN/m (K(0.5)), was very sensitive to pH. The maximal increment in pi upon drug incorporation into the monolayer (deltapi(max)) will depend on the phospholipid collapse pressure (pi(c)), the monolayers's compressibility and drug's size, shape and charge. The higher pi(c) of dpPC lead to higher pi(cut-off) values (maximal pi allowing drug penetration), if compared with dpPA. In dpPC and dpPA pi(cut-off) decreased as a function of the molecular size of the uncharged drugs. In dpPA, protonated drugs became electrostatically trapped at the monolayer surface hence drug penetration, monolayer deformation and pi increase were impaired and the correlation between pi(cut-off) and drug molecular size was lost.     

Identification of a human stomach alcohol dehydrogenase with distinctive kinetic properties

A new form of alcohol dehydrogenase, designated mu-alcohol dehydrogenase, was identified in surgical human stomach mucosa by isoelectric focusing and kinetic determinations. This enzyme was anodic to class I (alpha, beta, gamma) and class II (pi) alcohol dehydrogenases on agarose isoelectric focusing gels. The partially purified mu-alcohol dehydrogenase, specifically using NAD+ as cofactor, catalyzed the oxidation of aliphatic and aromatic alcohols with long chain alcohols being better substrates, indicating a barrel-shape hydrophobic binding pocket for substrate. mu-Alcohol dehydrogenase stood out in high Km values for both ethanol (18 mM) and NAD+ (340 microM) as well as in high Ki value (320 microM) for 4-methylpyrazole, a competitive inhibitor for ethanol. mu-Alcohol dehydrogenase may account for up to 50% of total stomach alcohol dehydrogenase activity and appeared to play a significant role in first-pass metabolism of ethanol in human.     

Active site mechanics of liver microsomal cytochrome P-450

For a set of 10 para-substituted toluene derivatives, three enzymatic constants were determined describing their interaction with purified rabbit liver microsomal P-450LM2. The three constants were the catalytic rate constant (Kcat) for hydroxylation, the apparent dissociation constant (Kd) for the enzyme-substrate complex, and the interaction energy (delta Gint) between the substrate-binding and spin-state equilibria. The para-substituents of the toluene substrates were: hydrogen, fluoro, bromo, chloro, iodo, nitro, methyl, cyano, isopropyl, and t-butyl. Linear free energy correlations were sought between the enzymatic constants and several physical constants of the individual substrate molecules. These correlations would be useful both for empirical prediction purposes and for insight into active site chemistry and mechanics. Catalytic rates were correlated by a linear combination of the Hansch pi hydrophobic constant and the Hammett sigma value. A deuterium isotope effect (DV) of 2.6 for d8-toluene compared to d0-toluene confirmed that hydrogen abstraction was partially rate-limiting with this series of substrates. Apparent dissociation constants were predicted by a linear combination of the molar volume and pi, while the spin-state interaction energies were best predicted by a linear combination of the Hansch pi hydrophobic constant and the reciprocal of the dielectric constant.     

Edge-to-face CH/pi interaction between ligand Phe-phenyl and receptor aromatic group in the thrombin receptor activation

In the ligand/receptor interaction, the side chain phenyl group of phenylalanine (Phe) is involved in a so-called hydrophobic interaction, in which the Phe-phenyl group functions as a p element or merely as a hydrophobic element. The thrombin receptor-tethered ligand SFLLRNP consists of the Phe-2 residue essential for receptor activation. In order to explore the molecular characteristics of this Phe-2-phenyl group, a complete set of S/Phe/LLRNP peptides comprising six different difluorophenylalanine isomers [(F(2))Phe] was newly synthesized and assayed to evaluate their ability to induce the aggregation of human platelets. The assay results clarified several important structural elements to conclude that Phe-2-phenyl of S/Phe/LLRNP is in the edge-to-face CH/pi interaction with the receptor aromatic group, utilizing the Phe-phenyl edge along with adjacent benzene hydrogens at positions (2-3) or (5-6). It was also found that the fluorine atom at position 4 increases the acidity of the hydrogen mainly at its ortho position, resulting in a reinforcement of the CH/pi interaction and thus in an enhancement of biological activity. The H-->F replacement in the benzene ring was found to provide an effective structural examination to the Phe residue; i.e., to identify the hydrogens in the CH/pi interaction, and to strengthen the CH/pi interaction.     

Probing the peripheral anionic site of acetylcholinesterase with quantitative structure activity relationships for inhibition by biphenyl-4-acyoxylate-4'-N-Butylcarbamates

Biphenyl-4-acyoxylate-4'-N-butylcarbamates 1-8 are synthesized from 4,4'-biphenol and are characterized as the pseudosubstrate inhibitors of acetylcholinesterase. In other words, the inhibitors bind to the enzyme and react with the enzyme to form the tetrahedral intermediates for the K(i) steps, and then the tetrahedral intermediates exclude the leaving groups to form a common N-butycarbamyl enzyme intermediate for the k(c) steps. Due to a linear character of the 4,4'-biphenyl moiety, the 4'-N-butylcarbamate moieties of the inhibitors react with the Ser200 residue of the enzyme while the 4-acyoxylate moieties of the inhibitors, on the other hand, should fit in the peripheral anionic site of the enzyme, which is located at the mouth of the deep active site gorge. Thus, carbamates with varied acyl substituents at the 4-position of the biphenyl ring are good candidates for probing the quantitative structure activity relationships for the peripheral anionic site of the enzyme. The fact that the pK(i), log k(c), and log K(i) values are correlated with neither the Taft substituent constant (sigma*) nor the Taft steric constant (E(s)) indicates that the 4-acyoxylate moieties of the inhibitors are too far away from the reaction center. However, the pK(i), log k(c), and log K(i) values are linearly correlated with the Hansch hydrophobicity constant, pi. The intensity constants (psi) for these correlations are 0.16, -0.035, and 0.13, respectively. These results indicate that interactions between the 4-acyoxylate groups of the inhibitors and the peripheral anionic site of the enzyme are mainly hydrophobic ones. The correlation results are slightly improved by using the two-parameter correlations with the Taft substituent steric constant, E(s), and pi. For pK(i), log k(c), and log K(i)-E(s)-pi correlations, the psi values are 0.21, -0.021, and 0.19, respectively; the intensity constants for steric effect (delta) are 0.08, 0.022, and 0.10, respectively. Besides hydrophobic interactions, the two-parameter correlations also suggest that little steric hindrance occurs for the bulkier inhibitors to pass by the peripheral anionic site of the enzyme.     

Hydrophobic nature of the active site of mandelate racemase

Mandelate racemase (EC 5.1.2.2) from Pseudomonas putida catalyzes the interconversion of the two enantiomers of mandelic acid with remarkable proficiency, stabilizing the altered substrate in the transition state by approximately 26 kcal/mol. We have used a series of substrate analogues (glycolates) and intermediate analogues (hydroxamates) to evaluate the contribution of the hydrophobic cavity within the enzyme's active site to ligand binding. Free energy changes accompanying binding of glycolate derivatives correlated well with the hydrophobic substituent constant pi and the van der Waals surface areas of the ligands. The observed dependence of the apparent binding free energy on surface area of the ligand was -30 +/- 5 cal mol(-1) A(-2) at 25 degrees C. Free energy changes accompanying binding of hydroxamate derivatives also correlated well with pi values and the van der Waals surface areas of the ligands, giving a slightly greater free energy dependence equal to -41 +/- 3 cal mol(-1) A(-2) at 25 degrees C. Surprisingly, mandelate racemase exhibited a binding affinity for the intermediate analogue benzohydroxamate that was 2 orders of magnitude greater than that predicted solely on the basis of hydrophobic interactions. This suggests that there are additional specific interactions that stabilize the altered substrate in the transition state. Mandelate racemase was competitively inhibited by (R,S)-1-naphthylglycolate (apparent K(i) = 1.9 +/- 0.1 mM) and (R,S)-2-naphthylglycolate (apparent K(i) = 0.52 +/- 0.03 mM), demonstrating the plasticity of the hydrophobic pocket. Both (R)- (K(m) = 0.46 +/- 0.06 mM, k(cat) = 33 +/- 1 s(-1)) and (S)-2-naphthylglycolate (K(m) = 0.41 +/- 0.03 mM, k(cat) = 25 +/- 1 s(-1)) were shown to be alternative substrates for mandelate racemase. These kinetic results demonstrate that no major steric restrictions are imposed on the binding of this bulkier substrate in the ground state but that steric factors appear to impair transition state/intermediate stabilization. 2-Naphthohydroxamate was identified as a competitive inhibitor of mandelate racemase, binding with an affinity (K(i) = 57 +/- 18 microM) that was reduced relative to that observed for benzohydroxamate and that was in accord with the approximately 10-fold reduction in the value of k(cat)/K(m) for the racemization of 2-naphthylglycolate. These findings indicate that, for mandelate racemase, steric constraints within the hydrophobic cavity of the enzyme-intermediate complex are more stringent than those in the enzyme-substrate complex.     

Influence of hydrophobic and steric effects in the acyl group on acylation of alpha-chymotrypsin by N-acylimidazoles

The second-order rate constants k2/Km for acylation of alpha-chymotrypsin by a series of N-acylimidazole derivatives of aliphatic carboxylic acids have been determined at 30 degrees C by proflavin displacement from the active site. With cyclohexyl-substituted N-acylimidazoles, the rate constants increase with increasing chain length of the acyl group; i.e., k2/Km is in the order cyclohexylcarbonyl less than cyclohexylacetyl less than beta-cyclohexylpropionyl. The latter substrate has k2/Km = 1.2 X 10(6) M-1 s-1 at pH 8.0, which appears to be a maximum value for N-acylimidazole substrates. A further increase in the chain length of the acyl group with (gamma-cyclohexylbutyryl)imidazole results in a decrease in k2/Km. Hydrophobic effects of the hydrocarbon acyl groups are of predominant importance with regard to the relative values of k2/Km for aliphatic N-acylimidazole substrates. There is a linear correlation of the logarithms of the rate constants at pH 8.0 with the hydrophobic substituent constants, pi, having a slope of 1.71 (r = 0.90). On the other hand, there is little apparent correlation with the Taft steric effect constants, Es. A four-parameter equation including both pi and Es improved the correlation only slightly [log (k2/Km) = 1.88 pi + 1.01 Es + C]. In contrast, steric effects as reflected in the Es constants are the major influence in acylation of the enzyme by corresponding p-nitrophenyl esters. There are very likely significant differences in transition-state structure with the two types of substrates.     

The importance of CH/pi interactions to the function of carbohydrate binding proteins

It is suggested that the interactions between the hydrophobic C-H groups of carbohydrate residues and the pi-electron systems of aromatic amino-acid residues play an important role in the ligand-recognition function of carbohydrate-binding proteins. This review focuses on our recent structural and functional studies of human lysozyme and hevein-domain type lectins (wheat-germ agglutinin and Ac-AMP2) aimed at understanding how CH/pi interactions are involved in the actual binding events.     

Characterization of toad liver glutathione transferase

The major form of glutathione transferase from the toad liver previously designed as Bufo bufo liver GST-7.6 (A. Aceto, B. Dragani, T. Bucciarelli, P. Sacchetta, F. Martini, S. Angelucci, F. Amicarelli, M. Miranda and C. Di Ilio, Biochem. J. 289 (1993) 417-422) has been characterized. According to its partial amino acid sequence, the toad enzyme may be included in the pi class GST and named bbGST P2-2. However, bbGST P2-2 appears to be immunologically, structurally and kinetically distinct from any other members of pi family, including bbGST P1-1, suggesting that it may constitute a subset of pi class GST. The data support the hypothesis that the transition from aquatic to terrestrial life causes a switch of the GST amphibian pattern promoting the expression of a GST form (bbGST P2-2) able to counteract, with higher efficiency, the toxic effects of reactive metabolites of oxidative metabolism and those of hydrophobic xenobiotics.     

Anion transport in red blood cells. I. Chemical properties of anion recognition sites as revealed by structure-activity relationships of aromatic sulfonic acids.

The present study is concerned with the chemical factors that determine the inhibitory properties of reversible aromatic sulfonic acids on sulfate exchange system of human red blood cells. Two series of compounds were tested for inhibitory potencies: benzene sulfonic acid (BS) and 2,2'-disulfonic stilbene (DS) derivatives, each series with substituent groups such as Cl, OH, NH2, NO2, NNN, N-acetamido, and N-benzoamido. As judged by various kinetic criteria, all congeners of BS and DS appear to have common sites of action in the anion transport system. The range of inhibitory potencies, as defined by the concentration required to produce 50% inhibition (ID50), varied over a 10(4) range (ID50:2-50,000 microM). The degree of inhibition was correlated with two physicochemical properties of the substituent groups: (a) lipophilicity, as judged by the pi values (Hansch factor) of the groups; and (b) the electronic character, as judged by sigma values (Hammett factor) of the groups. Optimal correlations were obtained with a linear combination of the two factors. Based on the above structure-activity relationships and on a comparison between the inhibitory properties of congeners of BS and DS, we suggest that the microenvironment of substrate recognition sites bears a positive multipolar character and possesses functionally essential groups with electron donor capacity embedded in a hydrophobic area.     

Quantitative structure-activity relationship study of iodinated analogues of trimetoquinol as highly potent beta 2-adrenoceptor ligands.

Trimetoquinol and its derivatives, reported to be highly potent beta 2-adrenoceptor (beta 2-AR) and site-selective thromboxane A2/prostaglandin H2 (TP) receptor ligands are subjected to quantitative structure-activity relationship (QSAR) study. From the significant correlation equation, obtained between the binding affinity, pKi (beta 2-AR) and the substitutional physicochemical parameters such as molar refraction, (MR), hydrophobic constant, (pi) and resonance parameter, (R), the receptor binding interactions associated with the varying sites of these compounds are discussed. The QSAR study has also explored the possibilities of having the analogous of improved binding affinities in future synthetic efforts. Likewise, the ratio of binding affinities, expressed as-log[Ki(beta 2-AR)/Ki alpha(TP)] related to two receptors are significantly correlated with MR and pi of the substituents and the relationship may, therefore, be helpful in developing the agents of greater selectivity on beta 2-AR versus TP receptor and vice versa.     

alpha-Tocopherol inhibits human glutathione S-transferase pi

alpha-Tocopherol is the most important fat-soluble, chain-breaking antioxidant. It is known that interplay between different protective mechanisms occurs. GSTs can catalyze glutathione conjugation with various electrophiles, many of which are toxic. We studied the influence of alpha-tocopherol on the activity of the cytosolic pi isoform of GST. alpha-Tocopherol inhibits glutathione S-transferase pi in a concentration-dependent manner, with an IC(50)-value of 0.5 microM. At alpha-tocopherol additions above 3 microM there was no GST pi activity left. alpha-Tocopherol lowered the V(max) values, but did not affect the K(m) for either CDNB or GSH. This indicates that the GST pi enzyme is noncompetitively inhibited by alpha-tocopherol. An inhibition of GST pi by alpha-tocopherol may have far-reaching implications for the application of vitamin E.     

Effects of a cationic and hydrophobic peptide, KL4, on model lung surfactant lipid monolayers.

We report on the surface behavior of a hydrophobic, cationic peptide, [lysine-(leucine)4]4-lysine (KL4), spread at the air/water interface at 25 degrees C and pH 7.2, and its effect at very low molar ratios on the surface properties of the zwitterionic phospholipid 1,2-dipalmitoylphosphatidylcholine (DPPC), and the anionic forms of 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) and palmitic acid (PA), in various combinations. Surface properties were evaluated by measuring equilibrium spreading pressures (pi(e)) and surface pressure-area isotherms (pi-A) with the Wilhelmy plate technique. Surface phase separation was observed with fluorescence microscopy. KL4 itself forms a single-phase monolayer, stable up to a surface pressure pi of 30 mN/m, and forms an immiscible monolayer mixture with DPPC. No strong interaction was detected between POPG and KL4 in the low pi region, whereas a stable monolayer of the PA/KL4 binary mixture forms, which is attributed to ionic interactions between oppositely charged PA and KL4. KL4 has significant effects on the DPPC/POPG mixture, in that it promotes surface phase separation while also increasing pi(e) and pi(max), and these effects are greatly enhanced in the presence of PA. In the model we have proposed, KL4 facilitates the separation of DPPC-rich and POPG/PA-rich phases to achieve surface refinement. It is these two phases that can fulfill the important lung surfactant functions of high surface pressure stability and efficient spreading.