Quantitative structure-activity relationship studies on benzodiazepine receptor binding: recognition of active sites in receptor and modelling of interaction.

A quantitative structure-activity relationship study was carried out for the binding of a series of 'classical' benzodiazepines (BZs) and some beta-carbolines with BZ receptors to investigate the active sites in the latter and the nature of the binding of compounds with them. Using the Hansch approach, an attempt was made to correlate binding affinities of compounds with various physico-chemical and electronic properties of substituents. The correlations obtained showed the main roles were played by the hydrophobic constant pi and the Hammett constant sigma (an electronic parameter) of various substituents. This led to the suggestion that BZ receptors have many additional hydrophobic, hydrogen bonding and polar sites other than those suggested by Hollinshead et al. (1990). From the present study, the Hollinshead model of interaction was found to be inadequate to account fully for the binding of all types of compounds.     

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.     

Binding of aromatic donor molecules to lactoperoxidase: proton NMR and optical difference spectroscopic studies

The interaction of aromatic donor molecules with lactoperoxidase (LPO) was studied using 1H-NMR and optical difference spectroscopy techniques. pH dependence of substrate proton resonance line-widths indicated that the binding was facilitated by protonation of an amino acid residue (with pKa of 6.1) which is presumably a distal histidine. Dissociation constants evaluated from both optical difference spectroscopy and 1H-NMR relaxation measurements were found to be an order of magnitude larger than those for binding to horse radish peroxidase (HRP), indicating relatively weak binding of the donors to LPO. The dissociation constants evaluated in presence of excess of I- and SCN- showed a considerable increase in their values, indicating that the iodide and thiocyanate ions compete for binding at the same site. The dissociation constant of the substrate binding was, however, not affected by cyanide binding to the ferric centre of LPO. All these results indicate that the organic substrates bind to LPO away from the ferric center. Comparison of the dissociation constants between the different substrates suggested that hydrogen bonding of the donors with the distal histidine amino acid, and hydrophobic interaction between the donors and the active site contribute significantly towards the associating forces. Free energy, entropy and enthalpy changes associated with the LPO-substrate equilibrium have been evaluated. These thermodynamic parameters were found to be all negative and relatively low compared to those for binding to HRP. The distances of the substrate protons from the ferric center were found to be in the range 9.4-11.1 A which are 2-3 A larger than those reported for the HRP-substrate complexes. These structural informations suggest that the heme in LPO may be more deeply buried in the heme crevice than that in the HRP.     

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.     

HPTLC chromatography of androstene derivates. Application of normal phase thin-layer chromatographic retention data in QSAR studies

The chromatographic behavior of seven 16-oximino derivatives of 3beta-hydropxy-5-androstene have been investigated using the normal-phase (NP) HPTLC chromatographic mode of the type silica-non-polar diluent (benzene)-polar modifier (acetonitrile, ethyl acetate, or dioxane). The linear relationship between the retention constants (R(M)) and the logarithm of the organic modifier content in the mobile phase allowed for the calculation of R(M)0 values. The influence of substituent in the molecule on extrapolated retention data is discussed. To better understand the retention mechanism in the separation of androstene compounds, the functional group contributions (tauX) were compared with Hansch substituent constants (pi). An attempt to quantitate the lipophilicity of the investigated compounds using normal phase thin-layer chromatographic R(M)0 value was made. Also, the relative lipophilicity values determined previously by RPC as well as activity were compared with NPC data.     

Alcoholysis catalyzed by Candida antarctica lipase B in a gas/solid system: effects of water on kinetic parameters

The influence of water on the kinetics of alcoholysis of methyl propionate and n-propanol catalyzed by immobilized lipase B from Candida antarctica was studied in a continuous solid/gas reactor. In this reactor, the solid phase is composed of a packed enzymatic sample which is percolated by gaseous nitrogen, simultaneously carrying gaseous substrates to the enzyme while removing reaction products. In this system, interactions between the enzyme and nonreacting molecules are avoided, since no solvent is present, and it is thus more easy to assess the role of water. To this end, alcohol inhibition constant, substrates dissociation constants as well as acylation rate constant and ratio of acylation to deacylation rate constants have been determined as a function of water activity (a(w)). Data obtained highlight that n-propanol inhibition constant and dissociation constant of methyl propionate are a lot affected by a(w) variations whereas water has no significant effect on the catalytic acylation step nor on the ratio of acylation to deacylation rate constants. These results suggest the water-independent character of the transition step.     

Examining the role of hydrogen bonding interactions in the substrate specificity for the loading step of polyketide synthase thioesterase domains

The final step in polyketide synthase-mediated biosynthesis of macrocyclic polyketides is thioesterase (TE)-catalyzed cyclization of a linear polyketide acyl chain. TEs are highly specific in the chemistry they catalyze. Understanding the molecular basis for substrate specificity of TEs is crucial for engineering these enzymes to macrocyclize non-native linear substrates. We investigated the role of hydrogen bonding interactions in the substrate specificity of formation of an acyl-enzyme intermediate for the TE from the 6-deoxyerythronolide B biosynthetic pathway. Thirteen single site-directed mutants were constructed, via removal of side chain hydrogen bonding groups from the binding cavity. Specificity constants for four different substrates with and without hydrogen bond donors and acceptors were determined for the five active mutants. The relative magnitude of specificity constants for substrates did not change for the mutant TEs. Circular dichroism spectroscopy was used to show that the majority of the catalytically inactive mutants did not fold. Two mutations were identified that enabled mutant TEs to form a folded but catalytically inactive tertiary structure. Our data do not support a role for hydrogen bonding in mediating substrate specificity of bacterial polyketide synthase TEs. The highly conserved polar residues in the binding cavity appear to stabilize the unusual substrate channel, which passes through the enzyme. We propose that hydrophobic interactions between the binding cavity and substrate drive substrate specificity, as is seen in many protein-carbohydrate recognition events. This hypothesis is in agreement with high-resolution structural data for nonhydrolyzable acyl-enzyme intermediates from the picromycin TE.     

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.     

Substrate binding to the multidrug transporter MepA

MepA is a multidrug transporter from Staphylococcus aureus that confers multidrug resistance through the efflux of a wide array of hydrophobic substrates. To evaluate the ability of MepA to recognize different substrates, the dissociation constants for interactions between MepA and three of its substrates (acriflavine (Acr), rhodamine 6G (R6G), and ethidium (Et)) were measured. Given that MepA is purified in the presence of detergents and that its substrates are hydrophobic, we examined the effect of the detergent concentration on the dissociation constant. We demonstrate that all three substrates interact directly with the detergent micelles. Additionally, we find the detergent effect on the K_D value to be highly substrate-dependent. The K_D value for R6G is greatly influenced by the detergent, whereas the K_D values for Acr and Et are only modestly affected. The effect of the inactive D183A mutant on binding was also evaluated. The D183A mutant shows lower affinity toward Acr and Et.     

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.     

Kinetic investigation of the alpha-chymotrypsin-catalyzed hydrolysis of peptide-ester substrates. The relationship between the structure of the peptide moiety and reactivity.

A number of peptide-ester substrates of the general structure Ac-Lxn-...-Lx2-Lx1-OMe have been synthesized and their alpha-chymotrypsin-catalyzed hydrolysis studied. The kinetic analysis involved varying the concentration of substrate and methanol product, and measuring rates along the entire progression curve. For the dipeptide esters Ac-Lx2-Lx1-OMe and the amino-acid derivatives Ac-Lx1-OMe the following constants could be determined: the dissociation constant of the enzyme-substrate complex, KEA, both rate constants of the acylation step, k23 and k32, and the forward rate constant of the deacylation step, k31. For the tripeptide ester Ac-Ala-Ala-Tyr-OMe it appears that the rate constant for the dissociation of the enzyme-substrate complex, k21, is smaller than the rate constant for acylation, k23. Thus, for this substrate only the association and dissociation rate constants k12 and k21 could be determined and the values of k23, k32 and k31 only indirectly estimated. The influence of structural changes in the peptide moiety of the substrates on reactivity has been established by comparing the rate constants of appropriate pairs of substrates. It was found that the substrate reactivity, as measured by k23/KEA, increase with the number and strength of the secondary interactions in a manner consistent with the binding scheme which has been proposed on the basis of crystallographic studies. The effect of a particular interaction on k23 and on KEA is dependent on the nature of the other interactions. However, the effect of k23/KEA appears to be independent of the presence of the other interactions and therefore characteristic of that particular interaction. The results for these substrates are compared with those found previously for a series of peptide substrates of the structure Ac-Lxn-... Lx2-...-Lx1-Gly-NH2 which have the same acyl moiety as the peptide esters studied in this work.     

Differential ligand recognition by the Src and phosphatidylinositol 3-kinase Src homology 3 domains: circular dichroism and ultraviolet resonance Raman studies

Src homology 3 (SH3) domains recognize Pro-rich motifs using a hydrophobic cleft which contains several conserved aromatic residues. To investigate how aromatic residues contribute to ligand recognition, circular dichroism (CD) and 235 nm excited ultraviolet resonance Raman spectroscopies have been applied to Src and phosphatidylinositol 3-kinase (PI3K) SH3s. The CD analysis shows that Src SH3 binds to RPLPPLP (R-core) using aromatic residues with a dissociation constant (K(d)) of 10 microM. Moreover, intensity increases of the Trp and Tyr Raman bands suggest that the interaction is mediated by hydrophobic contacts and/or hydrogen bond formation with both Trp and Tyr residues. In the interaction of Src SH3 with VSLARRPLPPLP (VSL12) (K(d) 0.8 microM), Trp118 appears to form a strong hydrogen bond with VSL12, judging from significant intensity increases of the Trp Raman bands and the reported complex structure. In contrast, PI3K SH3 binds to R-core and VSL12 with lower affinities (K(d) 34 and 18 microM, respectively), and the interactions are suggested to be mediated mainly by hydrophobic contacts and/or hydrogen bond formation with Tyr residue(s). In the D21N mutant (Asp21 --> Asn) of PI3K SH3, whose hydrophobic cleft is deformed, Trp55 is shown to be responsible for the interaction with VSL12 by intensity increases of the Trp Raman bands. However, the affinity is severely decreased (K(d) 330 microM). These observations imply that SH3 domains associate with their ligands with distinct use of aromatic residues and that hydrogen bond formation with an SH3-conserved Trp residue in the well-ordered hydrophobic cleft is important for stable complex formation.     

X-ray structures of Torpedo californica acetylcholinesterase complexed with (+)-huperzine A and (-)-huperzine B: structural evidence for an active site rearrangement

Kinetic and structural data are presented on the interaction with Torpedo californica acetylcholinesterase (TcAChE) of (+)-huperzine A, a synthetic enantiomer of the anti-Alzheimer drug, (-)-huperzine A, and of its natural homologue (-)-huperzine B. (+)-Huperzine A and (-)-huperzine B bind to the enzyme with dissociation constants of 4.30 and 0.33 microM, respectively, compared to 0.18 microM for (-)-huperzine A. The X-ray structures of the complexes of (+)-huperzine A and (-)-huperzine B with TcAChE were determined to 2.1 and 2.35 A resolution, respectively, and compared to the previously determined structure of the (-)-huperzine A complex. All three interact with the "anionic" subsite of the active site, primarily through pi-pi stacking and through van der Waals or C-H.pi interactions with Trp84 and Phe330. Since their alpha-pyridone moieties are responsible for their key interactions with the active site via hydrogen bonding, and possibly via C-H.pi interactions, all three maintain similar positions and orientations with respect to it. The carbonyl oxygens of all three appear to repel the carbonyl oxygen of Gly117, thus causing the peptide bond between Gly117 and Gly118 to undergo a peptide flip. As a consequence, the position of the main chain nitrogen of Gly118 in the "oxyanion" hole in the native enzyme becomes occupied by the carbonyl of Gly117. Furthermore, the flipped conformation is stabilized by hydrogen bonding of Gly117O to Gly119N and Ala201N, the other two functional elements of the three-pronged "oxyanion hole" characteristic of cholinesterases. All three inhibitors thus would be expected to abolish hydrolysis of all ester substrates, whether charged or neutral.     

QSAR for phospholipase A2 inhibitions by 1-acyloxy-3-N-n-octylcarbamyl-benzenes

1-Acyloxy-3-N-n-octylcarbamyl-benzenes are potent reversible competitive inhibitors of Naja mocambique mocambique phospholipase A2 with the Ki values from 9.6 to 119 microM. The pKi values are correlated to both Taft substituent constant sigma* and Hansch hydrophobicity constant pi. The pre-steady state inhibition studies indicate that the pK(S) values for the first inhibition step are linearly correlated to sigma* alone with the rho* of -0.09 for this correlation. Thus, the first inhibition step may involve the insertion of the inhibitor to hepta-coordinated Ca2+ ion of the enzyme to form the octa-coordinated Ca2+ ion of the enzyme. The log(k2/k(-2)) values for the second inhibition step are linearly correlated to pi alone, and the psi value for this correlation is 0.13. Therefore, the second step inhibition step may involve the van der Waals' interaction between the acyl group of the inhibitor and Tyr 69 of the enzyme.     

Interaction of aromatic donor molecules with manganese(III) reconstituted horseradish peroxidase: proton nuclear magnetic resonance and optical difference spectroscopic studies

The interaction of aromatic donor molecules with manganese(III) protoporphyrin-apohorseradish peroxidase complex [Mn(III)HRP] was investigated by optical difference spectroscopy and relaxation rate measurements of 1H resonances of aromatic donor molecules (at 500 MHz). pH dependence of substrate proton resonance line-widths indicated that the binding was facilitated by protonation of an amino acid residue (with a pKa of 6.1), which is presumably distal histidine. Dissociation constants were evaluated from both optical difference spectroscopy and 1H-NMR relaxation measurements (pH 6.1). The dissociation constants of aromatic donor molecules were not affected by the presence of excess of I-, CN- and SCN-. From competitive binding studies it was shown that all these aromatic donor molecules bind to Mn(III)HRP at the same site, which is different from the binding site of I-, CN- and SCN-. Comparison of the dissociation constants between the different substrates suggests that hydrogen bonding of the donors with distal histidyl amino acid and hydrophobic interaction between the donors and active site contribute significantly towards the associating forces. Free energy, entropy and enthalpy changes associated with the Mn(III)HRP-substrate equilibrium have been evaluated. These thermodynamic parameters were found to be all negative. Distances of the substrate protons from the paramagnetic manganese ion of Mn(III)HRP were found to be in the range of 7.7 to 9.4 A. The Kd values, the thermodynamic parameters and the distances of the bound aromatic donor protons from metal center in the case of Mn(III)HRP were found to be very similar as in the case of native Fe(III)HRP.     

Papain hydrolysis of X-phenyl-N-methanesulfonyl glycinates: a quantitative structure-activity relationship and molecular graphics analysis

The hydrolysis of 32 X-phenyl-N-methanesulfonyl glycinates by papain was investigated. It was found that the variation in the Michaelis constants could be rationalized by the following correlation equation: log 1/Km = 0.61 pi '3 + 0.46 MR4 + 0.55 sigma + 2.00 with a correlation coefficient of 0.945. In this expression, pi '3 is the hydrophobic constant for the more lipophilic of the two possible meta substituents, MR4 is the molar refractivity of 4-substituents, and sigma is the Hammett constant summed for all substituents. Using this equation, we designed, synthesized, and successfully predicted Km for a new congener intended to maximize binding (1/Km). The interactions involved in enzyme-substrate binding, as characterized by the correlation equation, are interpreted using a computer-constructed color three-dimensional-graphics molecular model of the enzyme active site. The nonenzymatic hydrolysis (both acid and basic) of phenyl hippurates yield rate constants which are well correlated by Hammett equations; however, log k for both acid and alkaline hydrolysis are not linearly related to log 1/Km or log kcat/Km.     

15-Hydroxyprostaglandin dehydrogenase from human placenta, II. Steady state kinetics and influence of prostaglandin F2alpha analogues (author's transl)]

A complete initial rate analysis of the forward reaction catalyzed by 15-hydroxyprostaglandin dehydrogenase from human term placenta was carried out at pH 7.4 (100mM triethanolamine) with the substrates NAD, and the prostaglandins E1, E2 and F2alpha. The limiting Michaelis constants, the dissociation constants, and the limiting maximum velocities for these substrates were calculated by fitting the obtained data by weighted linear regression analysis to the complete rate equation. The product inhibition of the reaction by NADH and 15-oxoprostaglandin was studied and the inhibition constants were graphically determined. The initial rate and inhibition patterns obtained indicate that the reaction follows kinetically an ordered Bi Bi mechanism. The prostaglandin F2alpha analogues ICI 81,008 and ICI 79,939 were not utilized by the enzyme. With ICI 81,008 a slight inhibition of the enzymatic reaction with prostaglandin F2alpha was observed, whereas ICI 79,939 showed no effect. The results are discussed with respect to their possible biological significance.     

Basis for the anomalous effect of competitive inhibitors on the kinetics of hydrolysis of short-chain phosphatidylcholines by phospholipase A2.

The effect of four specific competitive inhibitors on the kinetics of hydrolysis of short-chain diacyl-sn-glycero-3-phosphocholines below their critical micelle concentrations was examined. The kinetics of hydrolysis of short-chain substrates dispersed as solitary monomers were generally consistent with the classical Michaelis-Menten formalism; i.e., hydrolysis began without any latency period, the steady-state rate was observed at higher substrate concentrations, the steady-state initial rate showed a linear dependence on the enzyme concentration, and the hyperbolic dependence of the initial rate on the substrate concentration could be described in terms of KM and Vmax parameters. The competitive nature of the inhibitors used in this study has been established by a variety of techniques, and the equilibrium dissociation constants for the inhibitors bound to the enzyme were measured by the protection method [Jain et al. (1991) Biochemistry 30, 7306-7317]. The kinetics of hydrolysis in the presence of competitive inhibitors could be described by a single dissociation constant. However, the value of the dissociation constant obtained under the kinetic conditions was comparable to that obtained by the protection method for the inhibitor-enzyme complex bound to a neutral diluent, rather than to the value of the dissociation constant obtained with solitary monomeric inhibitors and the enzyme in the aqueous phase. Spectroscopic methods showed that the effectively lower dissociation constant of an inhibitor bound to PLA2 at the interface is due to the stabilization of the enzyme-inhibitor complex by interaction with other amphiphiles present in the reaction mixture.(ABSTRACT TRUNCATED AT 250 WORDS)     

Circular dichroic evidence for regulation of enzymatic activity by nonsubstrate hydrophobic ligand on glutathione S-transferase P.

1-Anilinonaphthalene-8-sulfonic acid (ANS) noncompetitively inhibited enzyme activity of glutathione S-transferase P for both glutathione and 1-chloro-2,4-dinitrobenzene (Ki = 30 microM). Dissociation constant for ANS.GST-P complex calculated from the binding study was 15 microM. From the similar values of the inhibition constant and the dissociation constant, it was concluded that specific ANS binding caused the loss of enzyme activity. In the protein structural analysis by circular dichroism, the secondary structures remarkably changed by ANS binding in accordance with the decrease of enzymatic activities. The conformational change of the protein and the decrease in enzymatic activity were reversed by dissociation of ANS. This fact strongly suggested that the enzymatic activity was regulated by a nonsubstrate hydrophobic ligand.     

Interaction of steroids with adrenal cytochrome P-450 (P-450(17)alpha,lyase) in liposome membranes

Purified cytochrome P-450(17)alpha,lyase from guinea-pig adrenal microsomes, which catalyzes progesterone 17 alpha-hydroxylation and sequentially C17-C20 bond cleavage of the 17 alpha-hydroxyprogesterone, was successfully incorporated into liposomal membranes composed of only phosphatidylcholine or of a phospholipid mixture of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine at a molar ratio of 5:3:1. Although the purified P-450(17)alpha,lyase was readily converted into P-420 in the detergent-solubilized system without substrates, the P-450 embedded in the liposomal membranes was found to be quite stable without the substrates. Using the P-450(17)alpha,lyase-proteoliposomes, the interaction of steroids with P-450(17)alpha,lyase was studied for progesterone, 17 alpha-hydroxyprogesterone and androstenedione in the liposomal system by optical difference spectroscopy and by equilibrium dialysis. The partition coefficients of steroids between the aqueous phase and the liposomal membranes were determined by the equilibrium dialysis. They were about 1.4-1.6-times higher in phosphatidylcholine liposomes than in the liposomes of the lipid mixture. The dissociation constants of the P-450-steroid complexes were calculated from the apparent dissociation constants using the partition coefficients for the situation where the substrate-binding site faces the lipid phase of the membranes or where it faces the aqueous phase. The dissociation constant in the former case was not affected by the lipid composition. These results suggest that P-450(17)alpha,lyase might interact only with the substrates in the lipid phase of the liposomal membranes.