Polyethylene glycol-modified papain catalyzes peptide bond formation in benzene

Summary Papain modified with 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine (activated PEG₂) was soluble in benzene and retained the enzymic activity. Acid-amide bond formation by the modified enzyme proceeded efficiently in benzene; N-benzoyl-L-alanine alkylamides were synthesized from N-benzoyl-L-alanine methyl ester and various alkylamines, and N-benzoyl-L-alaninyl(oligo)leucine ethyl ester was formed from N-benzoyl-L-alanine methyl ester and L-leucine ethyl ester.     

Ester synthesis at extraordinarily low temperature of -3 degrees C by modified lipase in benzene

The lipoprotein lipase from Pseudomonas fluorescens was modified with 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine. The modified lipase in which 55% of the amino groups in the enzyme molecule were coupled with polyethylene glycol was found to be soluble in benzene and catalyzed the reactions of ester synthesis, ester exchange, aminolysis and ester hydrolysis in benzene. The modified lipase had an extraordinary temperature-dependency: enzymic activity for methyl laurate synthesis from methyl alcohol and lauric acid increased with decreasing temperature and attained the maximum at the extremely low temperature of -3 degrees C. The optimum temperature for hydrolysis of methyl laurate was as low as -4 degrees C.     

Ester synthesis catalyzed by polyethylene glycol-modified lipase in benzene

Lipoprotein lipase, which catalyzes hydrolysis of emulsified triglycerides or water-insoluble esters, was modified with 2,4-bis(o-methoxy-polyethylene glycol)-6-chloro-s-triazine(activated PEG2). The modified lipase, in which 55% of the total amino groups in the lipase molecule, was soluble in organic solvents such as benzene, toluene, chloroform and dioxane. The modified lipase could catalyze ester synthesis reaction in benzene. When very hydrophobic substrates of lauryl alcohol and stearic acid were used, the ester synthesis reaction proceeded efficiently in the transparent benzene solution with the maximum activity of approximate 5.0 mumoles/min/mg of protein. Ester exchange and aminolysis reactions were also conducted with the modified lipase in benzene.     

Polyethylene glycol-modified catalase exhibits unexpectedly high activity in benzene

Bovine liver catalase with molecular weight of 248,000, which consists of four subunits, was modified with 2,4-bis(o-methoxypolyethylene glycol)-6-chloro-s-triazine(activated PEG2). The modified catalase became soluble in organic solvents such as benzene by increasing the degree of modification of amino groups in the enzyme with activated PEG2. The enzymic activity of the modified catalase in benzene, in which 42% of the total amino groups were coupled with the modifier, was unexpectedly high in comparison with the activity of non-modified catalase in aqueous system. The absorption spectrum of the modified catalase in benzene showed the characteristic pattern of a haem protein with Soret band at 405 nm. The temperature-activity profile of the modified catalase in benzene was clarified and its activation energy was estimated to be 1900 cal/mol.     

Kinetics and specificity of serine proteases in peptide synthesis catalyzed in organic solvents

Initial rates of peptide-bond synthesis catalyzed by poly(ethylene glycol)-modified chymotrypsin in benzene were determined using high-performance liquid chromatography. Enzymatic synthesis of N-benzoyl-L-tyrosyl-L-phenylalanine amide from N-benzoyl-L-tyrosine ethyl ester and L-phenylalanine amide was found to obey Michaelis-Menten kinetics an to be consistent with a ping-pong mechanism modified by a hydrolytic branch. The catalytic activity of modified chymotrypsin was dependent on both water concentration and type of organic solvent, the highest synthesis rate being obtained in toluene. Since the chymotrypsin specificity in the organic phase was actually altered, the enzyme's apparent kinetic parameters were determined for different substrates and compared to those obtained with other serine proteases in benzene. Both N-benzoyl-L-tyrosine ethyl ester and N-alpha-benzoyl-L-lysine methyl ester were comparable acyl donors in benzene and the (kcat/Km)app value of modified chymotrypsin was only 10-fold smaller than that obtained with poly(ethylene glycol)-modified trypsin in the synthesis of N-alpha-benzoyl-L-lysyl-L-phenylalanine amide. The change in chymotrypsin specificity was also confirmed through the binding of trypsin inhibitors in benzene. The overall results suggest that hydrophobic bonding between the enzyme and its substrate should not be taken into account during catalysis in the organic phase. In general, if hydrophobic interactions are involved in the binding of substrates to the active site in aqueous media, the replacement of water by hydrophobic solvents will induce some change in enzyme specificity. Moreover, secondary residues of enzyme-binding sites may also exert a significant influence on specificity since, as observed in this study, chymotrypsin exhibited high affinity for cationic substrates and cationic inhibitors as well in apolar solvents.     

Retinyl esters synthesis by polyethylene glycol-modified lipase in benzene

Summary Using polyethylene glycol-modified lipase we have succeeded in synthesizing retinyl palmitate through ester exchange reaction between retinyl acetate and palmitic acid in a transparent benzene solution. The product had much lower peroxide value than the one obtained by a conventional organic synthesis. As much as 85% of a substrate, retinyl acetate, was converted to the product in a day at 25°C. We have also succeeded in ester synthesis of retinyl oleate with very small peroxide value, although both substrates have double bonds and tend to be oxidized easily.     

Terpene alcohol ester synthesis by polyethylene glycol-modified lipase in benzene

Summary Lipase fromPseudomonas fragi modified with polyethylene glycol was soluble and active in organic solvents such as benzene and chlorinated hydrocarbons. Using the modified lipase, terpene alcohol esters were synthesized with various combinations of terpene alcohols (citronellol, geraniol, farnesol and phytol) and carboxylic acids (acetic-, propionic-, n-butyric-, and valeric acids) in benzene at 25°C. The yield was generally very high.     

Polyethylene glycol derivative-modified cholesterol oxidase soluble and active in benzene

Cholesterol oxidase from Nocardia sp. was modified with a synthetic copolymer of polyoxyethylene allylmethyldiether (PEG) and maleic acid anhydride (MA anhydride), poly(PEG-MA anhydride). The modified cholesterol oxidase, in which 64% of the amino groups in the protein molecule were coupled to poly(PEG-MA), was soluble in organic solvents and catalyzed the oxidation reaction of cholesterol in benzene to form 4-cholesten-3-one with the enzymic activity of 0.6 mumol/min/mg protein. Using the modified cholesterol oxidase together with polyethylene glycol-modified peroxidase, coupled reactions shown below took place in Cholesterol + O2----4-Cholesten-3-one + H2O2 H2O2 + o-Phenylenediamine----H2O + Oxidized o-Phenylenediamine transparent benzene solution, not in an emulsified system. The oxidation of cholesterol was directly determined in benzene by measuring the absorbance of oxidized o-phenylenediamine at 490 nm.     

Polymerization of 10-hydroxydecanoic acid in benzene with polyethylene glycol-modified lipase

Summary A hydrophobic substrate, 10-hydroxydecanoic acid having two functional groups (–OH and –COOH) in the molecule, was polymerized by ester bond formation with the polyethylene glycol-modified lipase in a transparent benzene solution. The polymer of 10-hydroxydecanoic acid was linearly elongated under a quite mild condition.     

Enzymic hydration of benzene oxide: Assay and properties

A radiometric assay for epoxide hydratase using [¹⁴C]benzene oxide as substrate has been developed. The reaction product trans-1,2-[¹⁴C]dihydroxy-1,2-dihydrobenzene (benzene dihydrodiol) was separated from the other components by simple extraction of the unreacted substrate and phenol (a rearrangement product) into a mixture of light petroleum and diethyl ether followed by extraction of the benzene dihydrodiol into ethyl acetate. The product was then estimated by scintillation counting. Using this assay the enzymic hydration of benzene oxide and the possible existence of a microsomal epoxide hydratase with a greater specificity toward benzene oxide were reinvestigated. The sequence of activities of microsomes from various organs was liver > kidney > lung > skin, the pH optimum of enzymic benzene oxide hydration was about pH 9.0, which is similar to that of styrene oxide hydration and both activities were equally stable when liver microsomal fractions were stored. The effect of low molecular weight inhibitors upon the hydration of styrene and benzene oxide by liver microsomes was similar in some cases and dissimilar in others. However, all the dissimilarities could be explained without recourse to the hypothesis of the existence of a separate benzene oxide hydratase. During enzyme purification studies the activity toward benzene oxide was inhibited by the detergent used (cutscum) but was recovered when the detergent was removed. Solubilization without significant loss of activity was successful using sodium cholate. This allowed immunoprecipitation studies, which were performed using monospecific antiserum raised against homogeneous epoxide hydratase. The dose-response curves of the extent of precipitation of activity with increasing amounts of added antiserum were indistinguishable for benzene oxide and styrene oxide as substrate. At high antiserum concentrations precipitation was complete with both substrates. The findings, taken together, indicate the presence in rat liver microsomes of a single epoxide hydratase catalyzing the hydration of both styrene and benzene oxide or the presence of enzymes so closely related that these cannot be distinguished by any of the criteria tested.     

Hydroxyl radical-mediated, cytochrome P-450-dependent metabolic activation of benzene in microsomes and reconstituted enzyme systems from rabbit liver

The mechanism of benzene oxygenation in liver microsomes and in reconstituted enzyme systems from rabbit liver was investigated. It was found that the NADPH-dependent transformation of benzene to water-soluble metabolites and to phenol catalyzed by cytochrome P-450 LM2 in membrane vesicles was inhibited by catalase, horseradish peroxidase, superoxide dismutase, and hydroxyl radical scavengers such as mannitol, dimethyl sulfoxide, and catechol, indicating the participation of hydrogen peroxide, superoxide anions, and hydroxyl radicals in the process. The cytochrome P-450 LM2-dependent, hydroxyl radical-mediated destruction of deoxyribose was inhibited concomitantly to the benzene oxidation. Also the microsomal benzene metabolism, which did not exhibit Michaelis-Menten kinetics, was effectively inhibited by six different hydroxyl radical scavengers. Biphenyl was formed in the reconstituted system, indicating the cytochrome P-450-dependent production of a hydroxycyclohexadienyl radical as a consequence of interactions between hydroxyl radicals and benzene. The formation of benzene metabolites covalently bound to protein was efficiently inhibited by radical scavengers but not by epoxide hydrolase. The results indicate that the microsomal cytochrome P-450-dependent oxidation of benzene is mediated by hydroxyl radicals formed in a modified Haber-Weiss reaction between hydrogen peroxide and superoxide anions and suggest that any cellular superoxide-generating system may be sufficient for the metabolic activation of benzene and structurally related compounds.     

Synthesis of two sequential polypeptides by dispersion in benzene and their circular dichroism spectra in aqueous solution: Poly(L-glu-L-lys-L-ala-gly) and poly(L-ala-D-glu-L-lys-D-ala-gly)

The monomers γ-benzylglutamyl-ε-benzyloxycarbonyl-lysylalanylglycine pentachlorophenyl ester and alanyl-γ-benzyl-D -glutamyl-ε-benzyloxycarbonyllysyl-D -alanyl-glycine pentachlorophenyl ester, were polymerized in dilute solutions of dimethylform-amide (DMF) or as dispersions in the same volume of benzene. After deprotection with hydrogen bromide, the products were either chromatographed on Sephadex G-50 or dialyzed. The polymers derived from the polymerization in benzene were considerably larger than those from DMF. The results in benzene indicated that high monomer to solvent ratios are not necessary for the production of high-molecular-weight sequential polypeptides. Circular dichroism spectra of the polymers and monomers at neutral and acid pH indicated that poly(L -Glu-L -Lys-L -Ala-Gly) exists in a random coil configuration and poly(L -Ala-D -Glu-L -Lys-D -Ala-Gly) exists in a β conformation.     

Cytochromes P450 involved with benzene metabolism in hepatic and pulmonary microsomes

Benzene is an occupational hazard and environmental toxicant found in cigarette smoke, gasoline, and the chemical industry. The major health concern associated with benzene exposure is leukemia. The toxic effects of benzene are dependent on its metabolism by the cytochrome P450 enzyme system. Previous research has identified CYP2E1 as the primary P450 isozyme responsible for benzene metabolism at low concentrations, whereas CYP2B1 is involved at higher concentrations. Our studies using microsomal preparations from human, mouse, and rat indicate that CYP2E1 is the P450 isozyme primarily responsible for benzene metabolism in lung and in liver. CYP2B isozymes have little involvement in benzene metabolism by either lung or liver. Our results also indicate that isozymes of the CYP2F subfamily may play a role in benzene metabolism by lung.     

Hydrophobicity of benzene

The present work tries to clarify the molecular origin of the poor solubility of benzene in water. The transfer of benzene from pure liquid phase into water is dissected in two processes: transfer from gas phase to pure liquid benzene; and transfer from gas phase to liquid water. The two solvation processes are analyzed in the temperature range 5-100 degrees C according to Lee's Theory. The solvation Gibbs energy change is determined by the balance between the work of cavity creation in the solvent, and the dispersive interactions of the inserted benzene molecule with the surrounding solvent molecules. The purely structural solvent reorganization upon solute insertion proves to be a compensating process. The analysis shows that the work of cavity creation is larger in water than in benzene, whereas the attractive energetic interactions are stronger in benzene than in water; this scenario is true at any temperature. Therefore, both terms act in the same direction, contrasting the transfer of benzene from pure liquid phase into water and determining its hydrophobicity.     

Nucleotide sequencing and characterization of the genes encoding benzene oxidation enzymes of Pseudomonas putida.

The nucleotide sequence of the genes from Pseudomonas putida encoding oxidation of benzene to catechol was determined. Five open reading frames were found in the sequence. Four corresponding protein molecules were detected by a DNA-directed in vitro translation system. Escherichia coli cells containing the fragment with the four open reading frames transformed benzene to cis-benzene glycol, which is an intermediate of the oxidation of benzene to catechol. The relation between the product of each cistron and the components of the benzene oxidation enzyme system is discussed.     

Maleylacetoacetate cis-trans isomerase: One-step double cis-trans isomerization of monomethyl muconate and the enzyme's probable role in benzene metabolism

Maleylacetoacetate cis-trans isomerase together with glutathione has been found to isomerize cis-trans isomers of monomethyl muconate. Isomerization about a single double bond and concerted double isomerization of the diene unit occurs. In addition to the variations in substrate structure previously identified the current results demonstrate that a cis,cis diene skeleton and a conjugated ester function are accepted by the enzyme. The present work and the fiding of trans,trans-muconic acid in the urine of benzene-fed mice ([16.] Xenobiotica 15, 211) suggest that maleylacetoacetate cis-trans isomerase may be responsible for the geometrical isomerization. However, cis,cis-muconaldehydic acid rather than cis,cis-muconic acid is suggested to be the early intermediate in benzene metabolism capable of rapid enzyme-catalyzed cis-trans isomerization.     

Non-linear production of benzene oxide-albumin adducts with human exposure to benzene

Benzene is initially metabolized to benzene oxide, which either undergoes further metabolism or reacts with macromolecules including proteins. Previously reported levels of benzene oxide-albumin adducts (BO-Alb) are analyzed from 30 workers exposed to 0.2-302 ppm benzene and 43 controls from Shanghai, China. Although both exposed workers and controls had significant levels of BO-Alb in their blood, exposed subjects' adduct levels (GM=378 pmol/g protein) were much greater than those of controls (GM=115 pmol/g protein). When the natural logarithm of the BO-Alb level was regressed upon the natural logarithm of exposure among the 30 exposed subjects, a strong effect of benzene exposure was observed (R(2)=0.612; p<0.0001). Because the slope of the relationship between BO-Alb and benzene exposure was significantly less than one in log-space, we infer that production of benzene oxide was less than proportional to benzene exposure. Since benzene is a substrate for CYP2E1, these results are consistent with saturation of CYP450 metabolism. They indicate that deviations from linear metabolism began at or below benzene exposures of 10 ppm and that pronounced saturation was apparent at 40-50 ppm. To our knowledge, this is the first study to investigate the linearity of human metabolism of a carcinogen based upon protein adducts.     

Identification of human cell responses to benzene and benzene metabolites

Benzene is a common air pollutant and confirmed carcinogen, especially in reference to the hematopoietic system. In the present study we analyzed cytokine/chemokine production by, and gene expression induction in, human peripheral blood mononuclear cells upon their exposure to the benzene metabolites catechol, hydroquinone, 1,2,4-benzenetriol, and p-benzoquinone. Protein profiling showed that benzene metabolites can stimulate the production of chemokines, the proinflammatory cytokines TNF-alpha and IL-6, and the Th2 cytokines IL-4 and IL-5. Activated cells showed concurrent suppression of anti-inflammatory cytokine IL-10 expression. We also identified changes in global gene expression patterns in response to benzene metabolite challenges by using high-density oligonucleotide microarrays. Treatment with 1,2,4-benzenetriol resulted in the suppression of genes related to the regulation of protein expression and a concomitant activation of genes that encode heat shock proteins and cytochrome P450 family members. Protein and gene expression profiling identified unique human cellular responses upon exposure to benzene and benzene metabolites.     

Circadian variation in lipid peroxidation induced by benzene in rats

Time-dependent effect of benzene, a potent carcinogenic industrial solvent, on lipid peroxidaiton and associated mechanisms has been studied in liver and kidney of rats. Significant differences were observed in the values of urinary phenol, microsomal malondialdehyde, reduced glutathione (GSH) and cytochrome P4502E1 in rats treated with benzene in morning and evening hours. Higher were the values for urinary phenol and hepatic microsomal malondialdehyde in rats administered benzene in evening hours. Contrarily, higher were the values for GSH and cytochrome P4502E1 in rats treated with benzene in morning hours. Increased microsomal lipid peroxidation has been attributed to low GSH status, whereas increased phenol concentration could be related to low activity of cytochrome P4502E1 in the liver of rats in evening hours. It is concluded that circadian rhythmicity in hepatic drug metabolizing enzyme system and GSH contributes in toxicity of benzene. The results are important from occupational health point of view.