Conformations of adducts formed between the genotoxic benzo[a]pyrene-7,8-dione and 2′-deoxycytidine

Benzo[a]pyrene-7,8-dione (BPQ) is formed by the activation of benzo[a]pyrene(B[a]P), which is an environmental toxic substance that is easily exposed in daily life, due to P450/epoxide hydrolase, and is a substance that induces DNA deformation by forming adducts with DNA. In this study, to investigate the form of bonding between BPQ and DNA, the structures of adducts between BPQ and 2′-deoxycytidine were examined. To examine BPQ–dC adduct conformation, geometry optimization of a total of 16 structural isomers was performed using the density functional theory method. In the structures of BPQ–dC adducts, for the cis-form, the angle between BPQ and dC is nearly perpendicular; but for the trans-form, the bending angle is small. The trans-form had a larger energy gap between ground state and excited state than the cis-form, and had a smaller HOMO–LUMO gap than the cis-form. Therefore, it was found that the trans-form absorbs stronger light and has higher reactivity than the cis-form. Molecular electrostatic potential was calculated and analyzed. The calculated ESP contour map shows the electrophilic and nucleophilic regions of the molecule.     

Photo-regulated duplex- and triplex-formation of the modified DNA carrying azobenzene.

The duplex- and triplex- forming activity of oligonucleotide was photo-regulated by using the isomerization of azobenzene in the side chain. When the azobenzene was isomerized from the trans-form to the cis-form by photo-irradiation, the melting temperatures of the duplex and triplex between the oligonucleotide and its complementary counterpart were significantly lowered.     

Quantification of spiroether isomers and herniarin of different parts of Matricaria matricarioides and flowers of Chamaemelum nobile

A simple HPLC-PAD-MS method was established to quantitatively analyse two spiroether isomers (cis-en-yn-dicycloether and trans-en-yn-dicycloether) and the main coumarin, herniarin, in chamomile herbs, simultaneously. By using this method, the contents of these three compounds in the flowers of two chamomile species, Roman chamomile (Chamaemelum nobile) and pineapple weed (Matricaria matricarioides), as well as in different parts of pineapple weed, were investigated. It was found that the flowers of both herbs contained large amounts of cis-en-yn-dicycloether and trans-en-yn-dicycloether, with the trans-form being more abundant than the cis-form. The leaves of pineapple weed were found to have the highest concentration of cis-en-yn-dicycloether and herniarin than the other parts. HPLC-PAD-MS-guided isolation and identification of other constituents are also discussed.     

Escherichia coli cyclophilin B binds a highly distorted form of trans-prolyl peptide isomer.

Cyclophilins facilitate the peptidyl-prolyl isomerization of a trans-isomer to a cis-isomer in the refolding process of unfolded proteins to recover the natural folding state with cis-proline conformation. To date, only short peptides with a cis-form proline have been observed in complexes of human and Escherichia coli proteins of cyclophilin A, which is present in cytoplasm. The crystal structures analyzed in this study show two complexes in which peptides having a trans-form proline, i.e. succinyl-Ala-trans-Pro-Ala-p-nitroanilide and acetyl-Ala-Ala-trans-Pro-Ala-amidomethylcoumarin, are bound on a K163T mutant of Escherichia coli cyclophilin B, the preprotein of which has a signal sequence. Comparison with cis-form peptides bound to cyclophilin A reveals that in any case the proline ring is inserted into the hydrophobic pocket and a hydrogen bond between CO of Pro and Neta2 of Arg is formed to fix the peptide. On the other hand, in the cis-isomer, the formation of two hydrogen bonds of NH and CO of Ala preceding Pro with the protein fixes the peptide, whereas in the trans-isomer formation of a hydrogen bond between CO preceding Ala-Pro and His47 Nepsilon2 via a mediating water molecule allows the large distortion in the orientation of Ala of Ala-Pro. Although loss of double bond character of the amide bond of Ala-Pro is essential to the isomerization pathway occurring by rotating around its bond, these peptides have forms impossible to undergo proton transfer from the guanidyl group of Arg to the prolyl N atom, which induces loss of double bond character.     

Trypsin-catalysed synthesis of oligopeptide amides: comparison of catalytic efficiency among trypsins of different origin (bovine, Streptomyces griseus and chum salmon).

A procedure has been developed for the synthesis of oligopeptide amide using inverse substrates as acyl donors with amino acid amide instead of p-nitroanilide as acyl acceptor and trypsins of different origin (bovine, Streptomyces griseus and chum salmon trypsins) as the catalyst. The effectiveness of this procedure was demonstrated by the synthesis of a pentapeptide, Boc-[Leu5]-enkephalin amide, as a model compound. The method was the first enzymatic method shown to be successful at each successive coupling step for the synthesis of the oligopeptide. Bovine and chum salmon trypsins were superior to Streptomyces griseus trypsin as the catalyst.     

Protease-catalyzed peptide synthesis using inverse substrates: the influence of reaction conditions on the trypsin acyl transfer efficiency

Benzyloxycarbonyl-L-alanine p-guanidinophenyl ester behaves as a trypsin "inverse substrate," i.e., a cationic center is included in the leaving group instead of being in the acyl moiety. Using this substrate as an acyl donor, trypsin catalyzes the synthesis of peptide bonds that cannot be split by this enzyme. An optimal acyl transfer efficiency was achieved between pH 8 and 9 at 30 degrees C.The addition of as much as 50% cosolvent was shown to be of minor influence on the acyl transfer efficiency, whereas the reaction velocity decreases by more than one order of magnitude. The efficiency of H-Leu-NH(2) and H-Val-NH(2) in deacylation is almost the same for "inverse" and normal type substrates.     

Protein kinase C activation by cis-fatty acid in the absence of Ca2+ and phospholipids

Protein kinase C has been shown to be a phospholipid/Ca2+-dependent enzyme activated by diacylglycerol (Nishizuka, Y. (1984) Nature 308, 693-697; Nishizuka, Y. (1984) Science 225, 1365-1370). We have reported that unsaturated fatty acids (oleic acid and arachidonic acid) can activate protein kinase C independently of Ca2+ and phospholipid (Murakami, K., and Routtenberg, A. (1985) FEBS Lett. 192, 189-193). This study shows that other cis-fatty acids such as linoleic acid also fully activate protein kinase C in the same manner. None of the saturated fatty acids (C:4 to C:18) nor the detergents (sodium dodecyl sulfate and Triton X-100) tested here were as effective as oleic acid. Unlike oleic acid, these detergents strongly inhibited protein kinase C activity induced by Ca2+/phosphatidylserine (PS) and diacylglycerol. Lowering the critical micelle concentration of oleic acid by increasing ionic strength also strongly inhibited oleic acid activation of protein kinase C activity. Dioleoylphosphatidylserine activated protein kinase C effectively (Ka = 7.2 microM). On the other hand, dimyristoylphosphatidylserine, which contains saturated fatty acids at both acyl positions, failed to activate protein kinase C even in the presence of Ca2+. These observations suggest that: protein kinase C activation by free fatty acid is specific to the cis-form and is not due to their detergent-like action, cis-fatty acid activation is due to the direct interaction of protein kinase C with the monomeric form of cis-fatty acids and not with the micelles of fatty acids, and cis-fatty acids at acyl positions in PS are also important for Ca2+/PS activation of protein kinase C.     

The substrate specificity of trypsin. The interrelationship between the structure and reactivity for quasi-substrates, derivatives of O-alkylmethylphosphonic acid and carboxylic acids

The kinetics of trypsin phosphorylation by thioesters of O-n-alkylmethylphosphonic acids, and reactivation of corresponding phosphoryl enzymes as well as kinetics of trypsin-catalyzed hydrolysis of p-nitrophenylcarboxylates have been studied. The rate constants for phosphorylation and dephosphorylation of trypsin depend on hydrophobicity of non-polar fragments in both substrate series in the same degree. On the other hand, the deacylation rate constants for a series of acyl trypsins do not change significantly while the apparent Michaelis constants change consistently with variations of non-polar acyl substituent. The study of substrate specificity of trypsin in terms of the transition state theory has allowed to elucidate the basis for low reactivity of trypsin towards the quasisubstrates.     

Complete amino acid sequence of chicken liver acyl carrier protein derived from the fatty acid synthase

The acyl carrier protein domain of the chicken liver fatty acid synthase has been isolated after tryptic treatment of the synthase. The isolated domain functions as an acceptor of acetyl and malonyl moieties in the synthase-catalyzed transfer of these groups from their coenzyme A esters and therefore indicates that the acyl carrier protein domain exists in the complex as a discrete entity. The amino acid sequence of the acyl carrier protein was derived from analyses of peptide fragments produced by cyanogen bromide cleavage and trypsin and Staphylococcus aureus V8 protease digestions of the molecule. The isolated acyl carrier protein domain consists of 89 amino acid residues and has a calculated molecular weight of 10,127. The protein contains the phosphopantetheine group attached to the serine residue at position 38. The isolated acyl carrier protein peptide shows some sequence homology with the acyl carrier protein of Escherichia coli, particularly in the vicinity of the site of phosphopantetheine attachment, and shows extensive sequence homology with the acyl carrier protein from the uropygial gland of goose.     

The nature of differences in amidase and esterase activities of some acyltrypsins]

Specific trypsin substrates (esters, anilides, amides, peptides) were shown to accelerate deacetylation of monoacetylated trypsin. The amidase activity of monoacetyl-, monopropyonyl-, and tetraformyl-trypsin was not manifested if the amidase activity of native enzyme was suppressed in these preparations by the ester substrates (benzoylarginine ethyl ester or p-nitrophenyl acetate). Therefore the differences in the residual amidase and esterase activities of these acylated trypsin preparations found earlier did not contradict the universality of the acylenzyme mechanism. These differences are due to the strong deacylating effect of specific substrate in its complex with the enzyme modified with nonspecific acyl residue. The latter fact is suggested to be an experimental confirmation of the "induced fit" hypothesis.     

Mechanism-based isocoumarin inhibitors for trypsin and blood coagulation serine proteases: new anticoagulants

Trypsin, porcine pancreatic kallikrein, and several blood coagulation enzymes, including bovine thrombin, bovine factor Xa, human factor Xa, human plasma factor XIa, human plasma factor XIIa, and human plasma kallikrein, were inactivated by a number of substituted isocoumarins containing basic functional groups (aminoalkoxy, guanidino, and isothiureidoalkoxy). 3-Alkoxy-4-chloro-7-guanidinoisocoumarins were found to be the most potent inhibitors for the coagulation enzymes tested with kobsd/[I] values in the range of 10(3)-10(5) M-1 s-1. 4-Chloro-3-isothiureidoalkoxyisocoumarins show high inhibitory potency toward porcine pancreatic kallikrein, human plasma kallikrein, human factor XIa, human factor XIIa, and trypsin with kobsd/[I] values of the order of 10(4)-10(5) M-1 s-1. The inhibition of these serine proteases by the substituted isocoumarins are time dependent, and the inactivation of trypsin by 3-alkoxy-4-chloro-7-guanidinoisocoumarins and 7-amino-4-chloro-3-(3-isothiureidopropoxy)isocoumarin occured concurrently with the loss of the isocoumarin absorbance. The complex formed from inactivation of trypsin by these two types of inhibitors was very stable and regained less than 4% activity in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (pH 7.5) after 1 day at 25 degrees C and regained 8-45% activity upon addition of buffered 0.29 M hydroxylamine. Trypsin inactivated by other inhibitors regained full activity upon standing or addition of hydroxylamine. Thrombin inactivated by 3-alkoxy-4-chloro-7-guanidinoisocoumarins was also quite stable and only regained 9-15% activity under similar conditions. These results are consistent with a proposed mechanism, where serine proteases inactivated by aminoalkoxyisocoumarins or isothiureidoalkoxyisocoumarins form acyl enzymes that will deacylate upon standing or addition of hydroxylamine. However, the acyl enzymes formed from 3-alkoxy-4-chloro-7-guanidinoisocoumarins or 7-amino-4-chloro-3-(3-isothiureidopropoxy)-isocoumarin will decompose further, probably through a quinone imine methide, to give an irreversibly inactivated enzyme by reaction with an active-site nucleophile such as His-57. The quinone imine methide intermediate may also react with a solvent nucleophile to give an acyl enzyme that can be reactivated by hydroxylamine. The inhibitors 4-chloro-7-guanidino-3-methoxyisocoumarin and 4-chloro-3-ethoxy-7-guanidinoisocoumarin have been tested as anticoagulants in human plasma and were effective at prolonging the prothrombin time. However, they are unstable in plasma (t1/2 = 4-8 min), and their in vivo utility may be limited.     

Study of the specificity of human lysosomal glycolipid hydrolases using synthetic fluorogenic substrates

On the basis of o-acylamino-4-methylumbelliferon, a number of beta-galactosides and beta-glucosides have been synthesized. The fluorogenic compounds obtained differ by the length of acyl residues. 6- and 8-hexadecanoylamino-4-methylumbelliferyl-beta-D-galactopyranosides (6-HMGal and 8-HMGal) are shown to be substrates for human galactocerebroside-beta-D-galactosidase. 6-HMGal analogues with shorter acyl residues, octanoyl (OMGal) and butanoyl (BMGal), were cleaved by another type of beta-galactosidase, GM1-ganglioside-beta-galactosidase. It has been established that 6-hexadecanoylamino-4-methylumbelliferyl-beta-D-glucopyranoside (HMGlc) is cleaved by human and animal glucocerebrosidase much slower than its chromogenic analogue (HMGlc). OMGlc did not exceed HNGlc either, though it is cleaved by glucocerebrosidase faster than HMGlc.     

Comparative specificity of porcine pancreatic kallikrein and bovine pancreatic trypsin. Importance of interactions N-terminal to the scissible bond

Hydrolyses catalyzed by bovine pancreatic trypsin and porcine pancreatic kallikrein were studied using synthetic peptide substrates of the type E chi-L chi 2-L chi 1 decreases Y and E chi-L chi 3-L chi 2-L chi 1 decreases Y with L chi 1 = Arg defining the hydrolysis position (indicated by the arrow). The leaving moiety Y was -OCH3, -NH-C6H4-p-NO2 and -Ala-NH2. Insight into interactions occurring between the active site of the enzymes and the acyl moiety of the substrates was gained by studying the influence on hydrolysis rate of structural variation of residues L chi 2 and L chi 3. Parallel analyses of the hydrolyses of the ester, anilide, and peptide substrates having the same acyl moiety considerably facilitated the interpretation of the kinetic data. Trypsin, but not kallikrein, displayed high reactivity even with relatively short substrates. Ac-Ala-Arg-Ala-NH2, for example, was a better substrate for trypsin than for kallikrein by a factor of 1.3 X 10(4) in terms of kcat and 5.9 X 10(4) in terms of kcat/Km. Reactivity differences of such magnitude were related to two main differences in enzyme-substrate interactions: the interaction of the arginine side chain of the substrate with the specificity pocket of the enzyme is optimal for trypsin but poor for kallikrein and the number of hydrogen bonds formed by the enzyme with the backbone section of the substrate on both sides of the specific residue is larger in the case of trypsin. The latter difference is found to be related to the structure of amino-acid residue 192 which is glutamine in trypsin and methionine in kallikrein.     

Characterization Of The S' Subsite Specificity Of Trypsin

The S' subsite specificity of bovine trypsin has been studied by partitioning of o-nitrophenylsulfenyl-L-arginyl-trypsin (formed using o-nitrophenylsulfenyl-L-arginine alkyl esters as acyl donors) between various amino acid-derived nucleophiles and water. The data obtained from spectrophotometric measurements confirmed a preference of trypsin for arginine residues in the P'₁-position, which is less marked but quite similar to that of chymotrypsin. The amides of leucine, phenylalanine, methionine, threonine, lysine and valine are better for synthesis than the corresponding methyl esters, and show a moderate nucleophile efficiency, decreasing in that order. Amides of acidic amino acids and D-leucine were ineffective in forming the peptide bond, whereas norvaline amide and dipeptide amides lead to increased aminolysis.     

Comparison of Streptomyces griseus and bovine trypsin by active site analysis using fluorescent acyl groups

The preparation of fluorescence labeled acyl enzymes (Streptomyces griseus trypsin) was successfully carried out using specific trypsin substrates, 'inverse substrates'. The topographical analysis of the structures of the area around the active site was carried out by measuring the fluorescence spectra of the acyl enzyme preparations and these results were compared with those of bovine trypsin. It was found that the polarity of the active site vicinity at pH 5 was similar to that of bovine trypsin, whereas considerable differences were noticed at lower pH as a result of pH-induced transformation. Conformational changes of the active site induced by the interaction with the specific ligand were analyzed from the fluorescence spectra. In these responses the two enzymes were quite distinguishable. The two enzymes active sites were also different in the energy transfer experiments. The spatial arrangements of the catalytic residues relative to the intrinsic tryptophan residues were suggested to be substantially different for the two enzymes.     

Protease inhibitors, part 13: Specific, weakly basic thrombin inhibitors incorporating sulfonyl dicyandiamide moieties in their structure

A series of compounds has been prepared by reaction of dicyandiamide with alkyl/arylsulfonyl halides as well as arylsulfonylisocyanates to locate a lead for obtaining weakly basic thrombin inhibitors with sulfonyldicyandiamide moieties as the S1 anchoring group. The detected lead was sulfanilyl-dicyandiamide (K1 of 3 microM against thrombin, and 15 microM against trypsin), which has been further derivatized at the 4-amino group by incorporating arylsulfonylureido as well as amino acyl/dipeptidyl groups protected at the amino terminal moiety with benzyloxycarbonyl or tosylureido moieties. The best compound obtained (ts-D-Phe-Pro-sulfanilyl-dicyandiamide) showed inhibition constants of 9 nM against thrombin and 1400 nM against trypsin. pKa measurements showed that the new derivatives reported here do indeed possess a reduced basicity, with the pKa of the modified guanidine moieties in the range 7.9-8.3 pKa units. Molecular mechanics calculations showed that the preferred tautomeric form of these compounds is of the type ArSO2N=C(NH2) NH-CN, probably allowing for the formation of favorable interaction between this new anchoring group and the active site amino acid residue Asp 189, critical for substrate/inhibitor binding to this type of serine protease. Thus, the main finding of the present paper is that the sulfonyldicyandiamide group may constitute an interesting alternative for obtaining weakly basic, potent thrombin inhibitors, which bind with less affinity to trypsin.     

S' subsite mapping of serine proteases based on fluorescence resonance energy transfer.

A microassay based on fluorescence resonance energy transfer has been developed to determine the S' specificity of serine proteases. The protease-catalyzed acyl transfer from a fluorescing acyl donor ester to a P'1/P'2 variable hexapeptide library of nucleophiles labeled with a fluorescence quencher leads to an internally quenched peptide product and a fluorescent hydrolysis product. The amount of fluorescence quenching allows one to draw conclusions about the interaction of the nucleophile at the S' sites of the protease. o-Aminobenzoic acid and 3-nitrotyrosine were used as an efficient donor-acceptor pair for the resonance energy transfer. The P'1/P'2 variable hexapeptide library with the general structure H-Xaa-Ala-Ala-Ala-Tyr(NO2)-Gly-OH and H-Ala-Xaa-Ala-Ala-Tyr(NO2)-Gly-OH, where Xaa represents Arg, Lys, Met, Phe, Ala, Gly, Ser, Gln and Glu, was prepared by solid-phase synthesis. Investigations of the S' specificity of trypsin, chymotrypsin and trypsin variants show that this assay is a fast and sensitive screening method for S' subsite mapping of serine proteases and is suitable for a high throughput screening. The assay might be useful for the development of restriction proteases and the estimation of yields in enzymatic peptide synthesis.     

The enigmatic acyl carrier protein phosphodiesterase of Escherichia coli: genetic and enzymological characterization

The acyl carrier proteins (ACPs) of fatty acid synthesis are functional only when modified by attachment of the prosthetic group, 4'-phosphopantetheine (4'-PP), which is transferred from CoA to the hydroxyl group of a specific serine residue. Almost 40 years ago Vagelos and Larrabee reported an enzyme from Escherichia coli that removed the prosthetic group. We report that this enzyme, called ACP hydrolyase or ACP phosphodiesterase, is encoded by a gene (yajB) of previously unknown function that we have renamed acpH. A mutant E. coli strain having a total deletion of the acpH gene has been constructed that grows normally, showing that phosphodiesterase activity is not essential for growth, although it is required for turnover of the ACP prosthetic group in vivo. ACP phosphodiesterase (AcpH) has been purified to homogeneity for the first time and is a soluble protein that very readily aggregates upon overexpression in vivo or concentration in vitro. The purified enzyme has been shown to cleave acyl-ACP species with acyl chains of 6-16 carbon atoms and is active on some, but not all, non-native ACP species tested. Possible physiological roles for AcpH are discussed.     

Isolation and characterization of trypsin inhibitors from cowpea (Vigna unguiculata)

The trypsin inhibitor fraction from cowpea (Vigna unguiculata) has been purified and characterized. Although the total trypsin inhibitor as purified by affinity chromatography on immobilised trypsin was shown to be heterogeneous by gel electrophoresis and isoelectric focusing as well as by function, it was relatively homogeneous in MW (ca 17 000) on gel filtration. The total trypsin inhibitor was divided into inhibitors active against trypsin only and active against trypsin and chymotrypsin by affinity chromatography on immobilised chymotrypsin. The ‘trypsin-only’ inhibitor was the major component of the total trypsin inhibitor. It was shown by isoelectric focusing and gel electrophoresis to contain several isoinhibitors. Determination of the combining weight of this inhibitor and investigation of the complexes formed with trypsin by gel filtration indicated the presence of two protease binding sites per inhibitor molecule. The chymotrypsin/trypsin inhibitor was also shown to be composed of several isoinhibitors. On the basis of gel electrophoresis and gel filtration in dissociating and non-dissociating media both inhibitors were considered to be dimeric molecules with the subunits linked by disulphide bonds; this implies that the ‘trypsin-only’ inhibitor has one binding site per subunit.