Kinetic and thermodynamic study of the tetramerization equilibrium of phosphorylase b

The association equilibrium of phosphorylase b, induced by AMP and in the presence of Mg2+, has been shown to be a reversible process that follows second order and first order reversible rate laws in the direction of tetramerization and dimerization respectively, this fact being independent of temperature and of enzyme and AMP concentrations. Moreover, rate and equilibrium constants have been evaluated and their dependence on temperature and AMP concentration studied in this work. An important role that the existence of two classes of AMP binding sites per enzymatic subunit plays in the aggregation properties of the enzyme has also been emphasized. In the presence of 0.1 and 1 mM AMP (binding to the high affinity site), the values of the change in enthalpy, activation energy of dimerization and activation energy of tetramerization are: -36 kcal/mol, +36 kcal/mol, and 0 kcal/mol respectively. Binding of AMP to the low affinity site (10 mM AMP) yields significant changes in the self-association equilibrium, since the preceding parameters reach the following values: -18, +32, and +14 kcal/mol.     

Kinetic study of the activation process of frog epidermis pro-tyrosinase by trypsin

1. The rate of tyrosinase formation has been calculated by coupling the activatory process of frog epidermis pro-tyrosinase by trypsin to the oxidation of L-DOPA to dopachrome. Under certain conditions ([trypsin]/[pro-tyrosinase] greater than or equal to 300), the lag period of the coupled reactions, tau, is independent of trypsin concentration. 2. The specific rate constant of tyrosinase formation at different temperatures has been calculated, ranging from 0.025 sec-1, at 5 degrees C to 0.248 sec-1, at 30 degrees C. 3. Thermodynamic parameters of the activatory process (delta G not equal to = + 18.5 kcal/mol; delta H not equal to = + 14.8 kcal/mol; delta S not equal to = -12.4 e.u.; Ea = + 15.3 kcal/mol), have been determined by the study of the system at different temperatures. These values are characteristic for a normal chemical reaction. 4. From these kinetic data, the order of products formation in the proteolytic step, can be determined, active tyrosinase being the last product released.     

次氯酸与不饱和脂肪酸氧化机制和转化产物的理论研究

摘要 目的：揭示次氯酸与不饱和脂肪酸的氧化反应机制及转化产物。方法：运用Gaussian 16软件包,采用密度泛函方法M06-2X(D3),结合6-31+G(d)基组,在SMD液相水模型水平下进行计算。结果：次氯酸与单不饱和脂肪酸油酸的氧化反应是先形成氯鎓离子中间体,氯鎓离子再与水分子反应生成氯醇,第一步氯鎓离子的形成是控速步骤,其反应活化自由能~8 kcal/mol。环氧化合物和短链的醛是两种转化产物,前者由氯醇脱氯化氢而来,而后者由环氧化合物和氯醇通过系列与次氯酸根的反应而得到,生成它们的控速步骤的反应活化自由能分别为23 和24 kcal/mol。选取两个乙基为取代基的乙烯为油酸模型,其与次氯酸反应的活化自由能仅比油酸高1 kcal/mol。计算得到次氯酸与亚油酸、顺-9,反-11 亚油酸、梓树酸和花生四烯酸模型氧化反应生成氯醇的活化自由能分别是~10、13、16和14 kcal/mol。结论：氯鎓离子中间体机制是次氯酸与不饱和脂肪酸氧化反应的主要机制,反应的活化自由能通常低于15 kcal/mol,意味着此氧化反应动力学上容易发生。氧化产物氯醇能转化为环氧化合物和短链的醛,但活化自由能较高,约23和24 kcal/mol。选取距离双键3个碳以内的结构为不饱和脂肪酸模型,它能够很好地反映不饱和脂肪酸的反应活性。     

Structural flexibility of canonical 2'-deoxyribonucleotides in DNA-like conformations

Quantitative characteristics of structural flexibility of the DNA elementary monomer units -5'-deoxycytidylic, 5'-thymidylic, 5'-deoxyadenylic and 5'-deoxyguanylic acid molecules--have been calculated with original methods. Root-mean-square deviations from equilibrium for all conformational parameters, caused by nuclei thermal or quantum zero-point vibrations, have been found to lie within 4 degrees divided by 25 degrees at 0 K and 7 degrees divided by 50 degrees at 298 K and corresponding relaxed force constants--within 1 divided by 35 kcal/mol x rad(-2). Their values have been found to be sensitive to the molecule's conformation. It has been proven, that the torsion angle gamma is the most rigid one whereas relaxed force constants for all other conformational variables are lower and comparable to each other. The data obtained could serve for development of structural-dynamical models of the DNA.     

Peroxynitrite reductase activity of selenoprotein glutathione peroxidase: a computational study

The peroxynitrite reductase activity of selenoprotein glutathione peroxidase (GPx) has been investigated using density functional theory calculations for peroxynitrite/peroxynitrous acid (ONOO-/ONOOH) substrates through two different "oxidation" and "nitration" pathways. In the oxidation pathway for ONOO-, the oxidation of GPx and the subsequent formation of the selenenic acid (E-Se-OH) occur through a concerted mechanism with an energy barrier of 4.7 (3.7) kcal/mol, which is in good agreement with the computed value of 7.1 kcal/mol for the drug ebselen and the experimentally measured barrier of 8.8 kcal/mol for both ebselen and GPx. For ONOOH, the formation of the E-Se-OH prefers a stepwise mechanism with an overall barrier of 6.9 (11.3) kcal/mol, which is 10.2 (11.2) kcal/mol lower than that for hydrogen peroxide (H2O2), indicating that ONOOH is a more efficient substrate for GPx oxidation. It has been demonstrated that the active site Gln83 residue plays a critical role during the oxidation process, which is consistent with the experimental suggestions. The nitration of GPx by ONOOH produces a nitro (E-Se-NO2) product via either of two different mechanisms, isomerization and direct, having almost the same barrier heights. A comparison between the rate-determining barriers of the oxidation and nitration pathways suggests that the oxidation of GPx by ONOOH is more preferable than its nitration. It was also shown that the rate-determining barriers remain the same, 21.5 (25.5) kcal/mol, in the peroxynitrite reductase and peroxidase activities of GPx.     

Binding to tubulin of the colchicine analog 2-methoxy-5-(2', 3', 4'-trimethoxyphenyl)tropone. Thermodynamic and kinetic aspects

The thermodynamics and kinetics of the binding to tubulin of the colchicine analog 2-methoxy-5-(2', 3', 4'-trimethoxyphenyl) tropone (termed AC because it lacks the B-ring of colchicine) have been characterized by fluorescence techniques. The fluorescence of AC is weak in aqueous solution and is enhanced 250-fold upon binding to tubulin. The following thermodynamic values were obtained for the interaction at 37 degrees C: K = 3.5 X 10(5) M-1; delta G0 = -7.9 kcal/mol; delta H0 = -6.8 kcal/mol; delta S0 = 3.6 entropy units. The AC-tubulin complex is 1-2 kcal/mol less stable than the colchicine-tubulin complex. The change in fluorescence of AC was employed to measure the kinetics of the association process, and quenching of protein fluorescence was used to measure both association and dissociation. The association process, like that of colchicine, could be resolved into a major fast phase and a minor slow phase. The apparent second order rate constant for the fast phase was found to be 5.2 X 10(4) M-1 S-1 at 37 degrees C, and the activation energy was 13 kcal/mol. This activation energy is 7-11 kcal/mol less than that for the binding of colchicine to tubulin. The difference in activation energies can most easily be rationalized by a mechanism involving a tubulin-induced conformational change in the ligand ( Detrich , H. W., III, Williams, R. C., Jr., Macdonald, T. L., Wilson, L., and Puett , D. (1981) Biochemistry 20, 5999-6005). Such a change would be expected to have a small activation energy in AC because it possesses a freely rotating single bond in place of the B-ring of colchicine.     

Contribution of monosaccharide residues in heparin binding to antithrombin III

The importance of 3-O- and 6-O-sulfated glucosamine residues within the heparin octasaccharide iduronic acid(1)----N-acetylglucosamine 6-O-sulfate(2)----glucuronic acid(3)----N-sulfated glucosamine 3,6-di-O-sulfate(4)----iduronic acid 2-O-sulfate(5)----N-sulfated glucosamine 6-O-sulfate(6)----iduronic acid 2-O-sulfate(7)----anhydromannitol 6-O-sulfate(8) was determined by comparing with synthetic tetra- and penta-saccharides its ability to bind human antithrombin. The octasaccharide had an affinity for antithrombin of 1 X 10(-8) M (10.2 kcal/mol) measured by intrinsic fluorescence enhancement at 6 degrees C. The synthetic pentasaccharide, consisting of residues 2-6, had an affinity of 3 X 10(-8) M (9.6 kcal/mol). The same pentasaccharide, except lacking the 3-O-sulfate on residue 4, had an affinity of 5 X 10(-4) M (4.5 kcal/mol) measured by equilibrium dialysis. The tetrasaccharide, consisting of residues 2-5, bound antithrombin with an affinity of 5 X 10(-6) M (6.8 kcal/mol). The tetrasaccharide, consisting of residues 3-6, had an affinity of 5 X 10(-5) M (5.5 kcal/mol). Since the loss of either the 6-O-sulfated residue 2 or the 3-O-sulfate of residue 4 results in a 4-5 kcal/mol or a 40-50% loss in binding energy of the pentasaccharide, these two residues must be the major contributors to the binding and must be linked to the biologic activity of the octasaccharide.     

Computational selection of flavonoid compounds as inhibitors against SARS-CoV-2 main protease,RNA-dependent RNA polymerase and spike proteins: A molecular docking study

An outbreak of Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has been recognized as a global health concern. Since, no specific antiviral drug is proven effective for treatment against COVID-19, identification of new therapeutics is an urgent need. In this study, flavonoid compounds were analyzed for its inhibitory potential against important protein targets of SARS-CoV-2 using computational approaches. Virtual docking was performed for screening of flavonoid compounds retrieved from PubChem against the main protease of SARS-CoV-2 using COVID-19 docking server. The cut off of dock score was set to >?9 kcal/mol and screened compounds were individually docked against main protease, RNA-dependent RNA polymerase, and spike proteins using AutoDock 4.1 software. Finally, lead flavonoid compounds were subjected to ADMET analysis. A total of 458 flavonoid compounds were virtually screened against main protease target and 36 compounds were selected based on the interaction energy value >?9 kcal/mol. Furthermore, these compounds were individually docked against protein targets and top 10 lead compounds were identified. Among the lead compounds, agathisflavone showed highest binding energy value of ?8.4 kcal/mol against main protease, Albireodelphin showed highest dock score of ?9.8 kcal/mol and ?11.2 kcal/mol against RdRp, and spike proteins, respectively. Based on the high dock score and ADMET properties, top 5 lead molecules such as Albireodelphin, Apigenin 7-(6″-malonylglucoside), Cyanidin-3-(p-coumaroyl)-rutinoside-5-glucoside, Delphinidin 3-O-beta-D-glucoside 5-O-(6-coumaroyl-beta-D-glucoside) and (-)-Maackiain-3-O-glucosyl-6″-O-malonate were identified as potent inhibitors against main protease, RdRp, and spike protein targets of SARS-CoV-2. These all compounds are having non-carcinogenic and non-mutagenic properties. This study finding suggests that the screened compounds include Albireodelphin, Apigenin 7-(6″-malonylglucoside), Cyanidin-3-(p-coumaroyl)-rutinoside-5-glucoside, Delphinidin 3-O-beta-D-glucoside 5-O-(6-coumaroyl-beta-D-glucoside) and (-)-Maackiain-3-O-glucosyl-6″-O-malonate could be the potent inhibitors of SARS-CoV-2 targets.     

Molecular docking and in vitro analysis of phytoextracts from B. serrata for antibacterial activities

The bioactives of Boswellia serrata have a role in ulcer healing therapies. Eleven bioactive compounds were obtained by GC-MS among which Cholan-24-oic acid, 3,12-bis (acetyl oxy) has a high molecular weight of 490.6719 with a retention time of 26.729. Twenty wound samples were collected aseptically from the labs and hospitals in and around the Namakkal districts of Tamilnadu, India. The antibacterial potential of E.coli showed a maximum inhibition of 27 mm against Tetracycline at 30µg. The ethanolic extract of the B. serrata shows a susceptibility of 19mm towards E. coli at 60µg concentration in MIC. Molecular docking results show the binding energy of Cholan-24-oic acid, 3,12-bis(acetyloxy) -8.6 (kcal/mol) followed by Pyrene, hexadecahydro- -6.7 (kcal/mol), and 5(1H)- Azulenone, 2,4,6,7,8,8a-hexahydro-3,8-dimethyl-4-(1-methylethylidene)-, (8S-cis)- 6.4 (kcal/mol) for further consideration.     

NMR kinetic studies of the ionophore X-537A-mediated transport of manganous ions across phospholipid bilayers.

Nuclear magnetic resonance spectroscopy has been applied as a method for studying manganous ions transport across the membrane of phosphatidylcholine vesicles. The rates of the ionophore X-537A (lasalocid A)-mediated Mn2+ transport have been measured as a function of ionophore concentration, pH of the vesicle suspension, and temperature. The translocation was found to occur via a neutral complex composed of one manganous ion bound in two ionized X-537A molecules (Mn X2). The activation energy for the overall transport process was determined to be 22 +/- 5 kcal/mol. Also a pKa of 5.0 +/- 0.2 was determined for the ionophore acid dissociation equilibrium in the vesicle suspension.     

Self-association of lysozyme. Thermochemical measurements: effect of chemical modification of Trp-62, Trp-108, and Glu-35.

Heats of dilution and of saccharide binding for hen egg white lysozyme have been measured at 30 degrees, 0.1 ionic strength, and pH 7 over the range 3 to 95 mg of protein/ml. The concentration dependence of the apparent relative molar enthalpy of lysozyme derived from these results gives the thermodynamic parameters for the formation of an intermolecular contact in an indefinite (head-to-tail) self-association process as delta G 0 = -3.9 kcal/mol, delta H 0 = -6.4 kcal/mol, and delta S 0 = -8,3 e.u. Oxindolealanine-62-lysozyme does not undergo self-association reactions that can be detected calorimetrically. This derivative reacts with native lysozyme to form hybrid polymeric species with free energy and enthalpy of interaction similar to those for the polymers of native lysozyme. These results are consistent with the intermolecular contact in the self-assocaition of lysozyme being asymmetric (head-to-tail). The heat of dilution of the derivative of lysozyme in which Glu-35 is blocked as the ester with oxindolealanine-108 is like that observed for native lysozyme in acid solution and is independent of pH. The concentration difference spectrum that develops through self-association is of the shape expected for introduction of an indole chromophore into a charge-free region of the intermolecular contact. The foregoing results indicate that Glu-35 and Trp-62 are part of the contact, that perturbation of Trp-108 does not make a principle contribution to the concentration difference spectrum, and that no acid group other than Glu-35 is perturbed by self-association. There is a small change in the heat of (GlcNAc)3 binding over the range 0.005 to 0.034 M saccharide. These data give the value of -1 kcal/mol for the enthalpy change for formation of the 2:1 saccharide-enzyme complex (ES2) from ES and S.     

Kinetics and thermodynamics of the interaction of 5-fluoro-2'-deoxyuridylate with thymidylate synthase

Thymidylate synthase (TS), 5-fluorodeoxyuridylate (FdUMP), and 5,10-methylenetetrahydrofolate (CH2-H4folate) form a covalent complex in which a Cys thiol of TS is attached to the 6-position of FdUMP and the one-carbon unit of the cofactor is attached to the 5-position. The kinetics of formation of this covalent complex have been determined at several temperatures by semirapid quench methods. Together with previously reported data the results permit calculation of every rate and equilibrium constant in the interaction. Conversion of the noncovalent ternary complex to the corresponding covalent complex proceeds at a rate of 0.6 s-1 at 25 degrees C, and the dissociation constant for loss of CH2-H4folate from the noncovalent ternary complex is approximately 1 microM. Activation parameters for the formation of the covalent complex were shown to be Ea = 20 kcal/mol, delta G+ = 17.9 kcal/mol, delta H+ = 19.3 kcal/mol, and delta S+ = 0.005 kcal/(mol.deg). The equilibrium constant between the noncovalent and covalent ternary complexes is approximately 2 X 10(4), and the overall dissociation constant of CH2-H4folate from the covalent complex is approximately 10(-11) M. The conversion of the noncovalent ternary complex to the covalent adduct is about 12-fold slower than kcat in the normal enzymic reaction. However, because the dissociation constant for CH2-H4folate from the noncovalent ternary complex is about 10-fold lower than that from the TS-dUMP-CH2-H4folate Michaelis complex, the terms corresponding to kcat/Km are nearly equal. We propose that some of the intrinsic binding energy of CH2-H4folate may be used to facilitate formation of a 5-iminium ion intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of essential ionizable groups and evaluation of subsite affinities in the active site of beta-D-glucosidase F1 from a Streptomyces sp

Partial amino acid sequences, the essential ionizable groups directly involved in catalytic reaction, and the subsite structure of beta-D-glucosidase purified from a Streptomyces sp. were investigated in order to analyze the reaction mechanism. On the basis of the partial amino acid sequences, the enzyme seemed to belong to the family 1 of beta-glucosidase in the classification of glycosyl hydrolases by Henrissat (1991). Dependence of the V and Km values on pH, when the substrate concentration was sufficiently lower than Km, gave the values of 4.1 and 7.2 for the ionization constants, pKe1 and pKe2 of essential ionizable groups 1 and 2 of the free enzyme, respectively. When the dielectric constant of the reaction mixture was decreased in the presence of 10% methanol, the pKe1 and pKe2, values shifted to higher, to +0.60 and +0.35 pH unit, respectively. The findings supported the notion that the essential ionizable groups of the enzyme were a carboxylate group (-COO-, the group 1) and a carboxyl group (-COOH, the group 2). The subsite affinities Ai's in the active site were evaluated on the basis of the rate parameters of laminarioligosaccharides. Subsites 1 and 2 having positive Ai values (A1 was 1.10 kcal/mol and A2 was 4.98 kcal/mol) were considered to probably facilitate the binding of the substrate to the active site. However, the subsites 3 and 4 showed negative Ai values (A3 was -0.21 kcal/mol and A4 was -2.8 kcal/mol).     

In-silico analysis of Sirt2 from Schistosoma mansoni: structures,conformations, and interactions with inhibitors

Sirtuins are NAD+-dependent lysine deacetylases member of the class III HDAC family. These are demonstrated to be therapeutic targets in parasitic diseases like schistosomiasis. Observations suggested that sirtuin enzyme is necessary for the functionality of fe/male reproductive system, due to which SmSirt2 is treated as a potential therapeutic target. There are no structural and molecular features of SmSirt2 have been reported yet. In this study, homology modeling has been used to determine the three-dimensional features of the SmSITRT2. Further, structure validation has been performed by energy minimization and Ramachandran plot. Validated structures are further subjected to molecular docking and virtual screening to find the best lead molecules for downstream analysis. Ten lead molecules were selected while comparing virtual screening of hSirt2 and SmSirt2 both. These leads are further compared with AKG2 which is known inhibitor of hSirt2 (?8.8 kcal/mol). Out of selected 10 leads, four of them (ZINC23995485 (?9.5 kcal/mol), ZINC53298162 (?9.4 kcal/mol), ZINC70927268 (?10.0 kcal/mol), ZINC89878705 (?11.2 kcal/mol)) have shown better interaction with SmSirt2, in which ZINC89878705 (?11.2 kcal/mol) shows a more compact packing as compared to AKG2 and rest of ligands. These molecules could be further subject to in vitro study and model of SmSirt2 has been proposed for further structure-based drug design projects concerning sirtuins from Schistosoma mansoni.     

A density functional investigation of the extradiol cleavage mechanism in non-heme iron catechol dioxygenases

The mechanism for extradiol cleavage in non-heme iron catechol dioxygenase was modelled theoretically via density functional theory. Based on the Fe(II)-His,His,Glu motif observed in enzymes, an active site model complex, [Fe(acetate)(imidazole)(2)(catecholate)(O(2))](-), was optimized for states with six, four and two unpaired electrons (U6, U4 and U2, respectively). The transfer of the terminal atom of the coordinated dioxygen leading to "ferryl" Fe=O intermediates spontaneously generates an extradiol epoxide. The computed barriers range from 19 kcal mol(-1) on the U6 surface to approximately 25 kcal mol(-1) on the U4 surface, with overall reaction energies of +11.6, 6.3 and 7.1 kcal mol(-1) for U6, U4 and U2, respectively. The calculations for a protonated process reveal the terminal oxygen of O(2) to be the thermodynamically favoured site but subsequent oxygen transfer to the catechol has a barrier of approximately 30-40 kcal mol(-1), depending on the spin state. Instead, protonating the acetate group gives a slightly higher energy species but a subsequent barrier on the U4 surface of only 7 kcal mol(-1) relative to the hydroperoxide complex. The overall exoergicity increases to 13 kcal mol(-1). The favoured proton-assisted pathway does not involve significant radical character and has features reminiscent of a Criegee rearrangement which involves the participation of the aromatic ring pi-orbitals in the formation of the new carbon-oxygen bond. The subsequent collapse of the epoxide, attack by the coordinated hydroxide and final product formation proceeds with an overall exoergicity of approximately 75 kcal mol(-1) on the U4 surface.     

The denaturation of alpha,beta and psi bovine trypsin at pH 3.0: evidence of intermediates

The conformational stability and the folding process of alpha, beta and Psi bovine trypsin at pH 3.0 followed by circular dichroism (CD) and size exclusion in HPLC have been analyzed as a function of urea concentration. The thermodynamic stability for a and b are deltaG = 15.91 +/- 0.28 kcal/mol, deltaG = 15.54 +/- 2.39 kcal/mol. respectively, and y trypsin is deltaG = 16.10 +/- 2.51 kcal/mol. The transition curves for alpha, beta and Psi forms suggest a molten globule state.     

Two mechanisms of H+/OH- transport across phospholipid vesicular membrane facilitated by gramicidin A.

Two rate-limiting mechanisms have been proposed to explain the gramicidin channel facilitated decay of the pH difference across vesicular membrane (delta pH) in the pH region 6-8 and salt (MCI, M+ = K+, Na+) concentration range 50-300 mM. 1) At low pH conditions (approximately 6), H+ transport through the gramicidin channel predominantly limits the delta pH decay rate. 2) At higher pH conditions (approximately 7.5), transport of a deprotonated species (but not through the channel) predominantly limits the rate. The second mechanism has been suggested to be the hydroxyl ion propogation through water chains across the bilayer by hydrogen bond exchange. In both mechanisms alkali metal ion transport providing the compensating flux takes place through the gramicidin channels. Such an identification has been made from a detailed study of the delta pH decay rate as a function of 1) gramicidin concentration, 2) alkali metal ion concentration, 3) pH, 4) temperature, and 5) changes in the membrane order (by adding small amounts of chloroform to vesicle solutions). The apparent activation energy associated with the second mechanism (approximately 3.2 kcal/mol) is smaller than that associated with the first mechanism (approximately 12 kcal/mol). In these experiments, delta pH was created by temperature jump, and vesicles were prepared using soybean phospholipid or a mixture of 94% egg phosphatidylcholine and 6% phosphatidic acid.     

NMR kinetic studies of the ionophore X-537A-mediated transport of manganous ions across phospholipid bilayers

Nuclear magnetic resonance spectroscopy has been applied as a method for studying manganous ions transport across the membrane of phosphatidylcholine vesicles. The rates of the ionophore X-537A (lasalocid A)-mediated Mn²⁺ transport have been measured as a function of ionophore concentration, pH of the vesicle suspension, and temperature. The translocation was found to occur via a neutral complex composed of one manganous ion bound to two ionized X-537A molecules (Mn X₂). The activation energy for the overall transport process was determined to be 22 ± 5 kcal/mol. Also a pK_a of 5.0 ± 0.2 was determined for the ionophore acid dissociation equilibrium in the vesicle suspension.     

Metabolism of myristicin by Depressaria pastinacella CYP6AB3v2 and inhibition by its metabolite

Although methylenedioxyphenyl (MDP) compounds, such as myristicin, are useful in the management of insecticide-resistant insects, the molecular mechanisms for their action in mammals and insects have not been elucidated. In this study, GC-MS analyses of methanol extracts of foliage of wild parsnip (Pastinaca sativa) have identified myristicin as a substrate for CYP6AB3v2, an imperatorin-metabolizing cytochrome P450 monooxygenase from Depressaria pastinacella (parsnip webworm). In contrast with its strong inhibitory effects on many mammalian P450s, myristicin is effectively metabolized by CYP6AB3v2 (V(max) and K(m) of 97.9 pmol/min/pmol P450 and 17.9 microM, respectively) at a rate exceeding that recorded previously for imperatorin, the only other known substrate for this highly specialized enzyme. The myristicin metabolite of CYP6AB3v2 is 1-(3',4'-methylenedioxy-5'-methoxyphenyl)-2,3-epoxypropane. Molecular dockings have indicated that, unlike other epoxide metabolites of furanocoumarins, this epoxide metabolite is likely to remain in the CYP6AB3v2 catalytic site due to its low binding energy (-31.0 kcal/mol). Inhibition assays indicate that myristicin acts as a mixed inhibitor of this insect P450 and suggest that the epoxide metabolite may be an intermediate involved in the formation of P450-methylenedioxyphenyl complexes.     

mRNA secondary structure in an open reading frame reduces translation efficiency in Bacillus subtilis.

The structural gene for thermostable neutral protease, nprM, has only one stacking region, whose energy is -16.3 kcal/mol (-68.2 kJ/mol). Mutations for increasing (-30.8 kcal/mol [128.9 kJ/mol] and decreasing (-5.0 kcal/mol [-20.9 kJ/mol]) the energy of the stacking region were introduced in nprM on the recombinant plasmid pMK1 by using site-directed mutagenesis without any amino acid substitutions. The resultant plasmids were designated pMK2 and pMK3, respectively. The enzyme productivity of the pMK2 carrier was about 40% lower than that of pMK1, whereas the productivity of the pMK3 carrier was about 5% higher. The higher the stability of the stacking regions, the lower the enzyme productivity that was observed. mRNA concentrations were almost the same in the cells harboring these three plasmids. These results indicate that the secondary structure of mRNA reduces the translation efficiency.