Substrate Report for: PropylparabenGeneral
Target
References: Search PubMed for references concerning: Propylparaben 2 more
Martinez-Martinez M, Coscolin C, Santiago G, Chow J, Stogios PJ, Bargiela R, Gertler C, Navarro-Fernandez J, Bollinger A and Ferrer M <32 more author(s)> Martinez-Martinez M, Coscolin C, Santiago G, Chow J, Stogios PJ, Bargiela R, Gertler C, Navarro-Fernandez J, Bollinger A, Thies S, Mendez-Garcia C, Popovic A, Brown G, Chernikova TN, Garcia-Moyano A, Bjergah GE, Perez-Garcia P, Hai T, Del Pozo MV, Stokke R, Steen IH, Cui H, Xu X, Nocek BP, Alcaide M, Distaso M, Mesa V, Pelaez AI, Sanchez J, Buchholz PCF, Pleiss J, Fernandez-Guerra A, Glockner FO, Golyshina OV, Yakimov MM, Savchenko A, Jaeger KE, Yakunin AF, Streit WR, Golyshin PN, Guallar V, Ferrer M (- 32) Ref: ACS Chemical Biology, 13:225, 2018 : PubMed ESTHER: Martinez-Martinez_2018_ACS.Chem.Biol_13_225 PubMedSearch: Martinez-Martinez 2018 ACS.Chem.Biol 13 225 PubMedID: 29182315 Gene_locus related to this paper: 9zzzz-a0a2k8jn75, 9zzzz-a0a2k8jt94, 9zzzz-a0a0g3fj44, 9zzzz-a0a0g3fh10, 9zzzz-a0a0g3fh03, 9bact-a0a1s5qkj8, 9zzzz-a0a0g3feh5, 9zzzz-a0a0g3fkz4, 9zzzz-a0a0g3fh07, 9zzzz-a0a0g3fh34, 9zzzz-a0a0g3fh31, 9bact-KY458167, alcbs-q0vqa3, 9bact-a0a1s5qki8, 9zzzz-a0a0g3feq8, 9zzzz-a0a0g3feh8, 9zzzz-a0a0g3fh19, 9bact-KY203037, 9bact-a0a1s5ql22, 9bact-a0a1s5qm34, 9bact-KY203034, 9bact-r9qzg0, 9bact-a0a1s5qly8, 9zzzz-a0a0g3fkz8, 9zzzz-a0a0g3feg9, 9zzzz-KY203033, 9zzzz-a0a0g3fes4, 9zzzz-a0a0g3fh42, 9bact-a0a1s5qlx2, 9zzzz-KY483651, 9bact-a0a1s5qmh4, 9zzzz-KY203032, 9zzzz-EH87, 9zzzz-a0a0g3fei1, 9zzzz-a0a0g3fet2, 9zzzz-KY483647, 9zzzz-EH82, 9zzzz-a0a0g3fe15, 9bact-KY203031, 9bact-t1w006, 9zzzz-a0a0g3fet6, 9bact-KY458164, geoth-g8myf3, 9bact-a0a1s5ql04, 9gamm-a0a1y0ihk7, 9bact-a0a1s5qly6, 9bact-a0a1s5qkg4, 9bact-a0a1s5qkm4, 9gamm-s5tv80, 9gamm-a0a0c4zhg2, 9zzzz-t1b379, 9gamm-KY483646, 9bact-KY458160, 9zzzz-a0a0g3fj57, 9gamm-s5t8349, 9arch-KY203036, 9bact-KY458168, 9zzzz-a0a0g3fes0, 9zzzz-t1be47, 9bact-KY458159, 9zzzz-a0a0g3fh39, 9bact-t1vzd5, 9prot-EH41, 9bact-Lip114, alcbs-q0vt77, 9bact-a0a1s5qke6, 9bact-a0a1s5qkf3, 9prot-SRP030024, 9gamm-s5t532, 9bact-a0a1s5qkl2, 9bact-a0a1s5qkk8, 9zzzz-KY203030, 9zzzz-t1d4I7, 9prot-KY019260, 9bact-a0a1s5qm38, 9arch-KY458161, 9prot-KY010302, 9zzzz-a0a0g3fl25, 9actn-KY010298, 9gamm-s5u059, 9bact-a0a1s5qmi7, 9bact-KY010297, 9bact-KY483642, 9bact-a0a1s5qkj1, 9bact-KY010299, 9bact-KY483648, alcbs-q0vtl7, 9bact-a0a1s5qf1, 9bact-a0a1s5qkg0, 9bact-a0a0h4tgu6, 9bact-MilE3, 9bact-LAE6, 9alte-MGS-MT1, 9bact-r9qzf7, 9gamm-k0c6t6, alcbs-q0vl36, alcbs-q0vlq1, alcbs-q0vq49, bacsu-pnbae, canar-LipB, canan-lipasA, geost-lipas, marav-a1u5n0, pseps-i7k8x5, staep-GEHD, symth-q67mr3, altma-s5cfn7, cycsp-k0c2b8, alcbs-q0vlk5, 9bact-k7qe48, 9bact-MGS-M1, 9bact-MGS-M2, 9bact-a0a0b5kns5, 9zzzz-a0a0g3fej4, 9zzzz-a0a0g3fj60, 9zzzz-a0a0g3fej0, 9zzzz-a0a0g3fj64, 9bact-a0a0b5kc16, 9zzzz-a0a0g3feg6, 9zzzz-a0a0g3feu6 Abstract Esterases receive special attention because their wide distribution in biological systems and environments and their importance for physiology and chemical synthesis. The prediction of esterases substrate promiscuity level from sequence data and the molecular reasons why certain such enzymes are more promiscuous than others, remain to be elucidated. This limits the surveillance of the sequence space for esterases potentially leading to new versatile biocatalysts and new insights into their role in cellular function. Here we performed an extensive analysis of the substrate spectra of 145 phylogenetically and environmentally diverse microbial esterases, when tested with 96 diverse esters. We determined the primary factors shaping their substrate range by analyzing substrate range patterns in combination with structural analysis and protein-ligand simulations. We found a structural parameter that helps ranking (classifying) promiscuity level of esterases from sequence data at 94% accuracy. This parameter, the active site effective volume, exemplifies the topology of the catalytic environment by measuring the active site cavity volume corrected by the relative solvent accessible surface area (SASA) of the catalytic triad. Sequences encoding esterases with active site effective volumes (cavity volume/SASA) above a threshold show greater substrate spectra, which can be further extended in combination with phylogenetic data. This measure provides also a valuable tool for interrogating substrates capable of being converted. This measure, found to be transferred to phosphatases of the haloalkanoic acid dehalogenase superfamily and possibly other enzymatic systems, represents a powerful tool for low-cost bioprospecting for esterases with broad substrate ranges, in large scale sequence datasets. Title: Inter-individual variability in esterases in human liver Jewell C, Bennett P, Mutch E, Ackermann C, Williams FM Ref: Biochemical Pharmacology, 74:932, 2007 : PubMed ESTHER: Jewell_2007_Biochem.Pharmacol_74_932 PubMedSearch: Jewell 2007 Biochem.Pharmacol 74 932 PubMedID: 17651701 Abstract Human liver has numerous hydrolytic enzymes involved in metabolism of endogenous and exogenous esters. Of these enzymes, carboxylesterases (EC 3.1.1.1) form an important group which hydrolyses many diverse ester substrates, including pro-ester drugs. Carboxylesterase activity was investigated in liver subcellular fractions from 22 individuals using the general carboxylesterase substrate phenylvalerate and the homologous series of esters methyl-, ethyl-, propyl-, butyl- and benzylparaben. The intra- and inter-individual variation in phenylvalerate and paraben metabolism was compared. Rates of hydrolysis were higher in microsomal fractions than cytosolic fractions for all compounds. The rate of paraben hydrolysis varied depending on the size of the paraben alcohol leaving group, showing a decrease with increasing leaving group size. Comparisons showed that individuals with high rates of hydrolysis towards methyl paraben also showed high rates of hydrolysis to the other parabens and phenylvalerate. Phenylvalerate as a non-specific carboxylesterase substrate was hydrolysed mainly by hCE1 in human livers and there was good correlation with small alcohol leaving group parabens, suggesting hCE1 involvement. Lower correlations with larger alcohol leaving group parabens are consistent with more hCE2 involvement. Title: Purification and characterization of PrbA, a new esterase from Enterobacter cloacae hydrolyzing the esters of 4-hydroxybenzoic acid (parabens) Valkova N, Lepine F, Labrie L, Dupont M, Beaudet R Ref: Journal of Biological Chemistry, 278:12779, 2003 : PubMed ESTHER: Valkova_2003_J.Biol.Chem_278_12779 PubMedSearch: Valkova 2003 J.Biol.Chem 278 12779 PubMedID: 12556461 Gene_locus related to this paper: entcl-PRBA Inhibitor(s) related to this paper: Tosyl-L-lysine-chloromethyl-ketone Abstract The esterase PrbA from Enterobacter cloacae strain EM has previously been shown to confer additional resistance to the esters of 4-hydroxybenzoic acid (parabens) to two species of Enterobacter. The PrbA protein has been purified from E. cloacae strain EM using a three-step protocol resulting in a 60-fold increase in specific activity. The molecular mass of the mature enzyme was determined to be 54,619 +/- 1 Da by mass spectrometry. It is highly active against a series of parabens with alkyl groups ranging from methyl to butyl, with K(m) and V(max) values ranging from 0.45 to 0.88 mM and 0.031 to 0.15 mM/min, respectively. The K(m) and V(max) values for p-nitrophenyl acetate were 3.7 mM and 0.051 mM/min. PrbA hydrolyzed a variety of structurally analogous compounds, with activities larger than 20% relative to propyl paraben for methyl 3-hydroxybenzoate, methyl 4-aminobenzoate, or methyl vanillate. The enzyme showed optimum activity at 31 degrees C and at pH 7.0. PrbA was able to transesterify parabens with alcohols of increasing chain length from methanol to n-butanol, achieving 64% transesterification of 0.5 mm propyl paraben with 5% methanol within 2 h. PrbA was inhibited by 1-chloro-3-tosylamido-4-phenyl-2-butanone and 1-chloro-3-tosylamido-7-amino-2-heptanone (TLCK), with K(i) values of 0.29 and 0.20 mM, respectively, and was irreversibly inhibited by Diisopropyl fluorophosphate (DFP) or diethyl pyrocarbonate. The stoichiometry of addition of DFP to the enzyme was 1:1 and only 1 TLCK molecule was found in TLCK-modified enzyme, as measured by mass spectrometry. Analysis of the tryptic digest of the DFP-modified PrbA demonstrated that the addition of a DFP molecule occurred at Ser-189, indicating the location of the active serine. 2 less
Martinez-Martinez M, Coscolin C, Santiago G, Chow J, Stogios PJ, Bargiela R, Gertler C, Navarro-Fernandez J, Bollinger A and Ferrer M <32 more author(s)> Martinez-Martinez M, Coscolin C, Santiago G, Chow J, Stogios PJ, Bargiela R, Gertler C, Navarro-Fernandez J, Bollinger A, Thies S, Mendez-Garcia C, Popovic A, Brown G, Chernikova TN, Garcia-Moyano A, Bjergah GE, Perez-Garcia P, Hai T, Del Pozo MV, Stokke R, Steen IH, Cui H, Xu X, Nocek BP, Alcaide M, Distaso M, Mesa V, Pelaez AI, Sanchez J, Buchholz PCF, Pleiss J, Fernandez-Guerra A, Glockner FO, Golyshina OV, Yakimov MM, Savchenko A, Jaeger KE, Yakunin AF, Streit WR, Golyshin PN, Guallar V, Ferrer M (- 32) Ref: ACS Chemical Biology, 13:225, 2018 : PubMed ESTHER: Martinez-Martinez_2018_ACS.Chem.Biol_13_225 PubMedSearch: Martinez-Martinez 2018 ACS.Chem.Biol 13 225 PubMedID: 29182315 Gene_locus related to this paper: 9zzzz-a0a2k8jn75, 9zzzz-a0a2k8jt94, 9zzzz-a0a0g3fj44, 9zzzz-a0a0g3fh10, 9zzzz-a0a0g3fh03, 9bact-a0a1s5qkj8, 9zzzz-a0a0g3feh5, 9zzzz-a0a0g3fkz4, 9zzzz-a0a0g3fh07, 9zzzz-a0a0g3fh34, 9zzzz-a0a0g3fh31, 9bact-KY458167, alcbs-q0vqa3, 9bact-a0a1s5qki8, 9zzzz-a0a0g3feq8, 9zzzz-a0a0g3feh8, 9zzzz-a0a0g3fh19, 9bact-KY203037, 9bact-a0a1s5ql22, 9bact-a0a1s5qm34, 9bact-KY203034, 9bact-r9qzg0, 9bact-a0a1s5qly8, 9zzzz-a0a0g3fkz8, 9zzzz-a0a0g3feg9, 9zzzz-KY203033, 9zzzz-a0a0g3fes4, 9zzzz-a0a0g3fh42, 9bact-a0a1s5qlx2, 9zzzz-KY483651, 9bact-a0a1s5qmh4, 9zzzz-KY203032, 9zzzz-EH87, 9zzzz-a0a0g3fei1, 9zzzz-a0a0g3fet2, 9zzzz-KY483647, 9zzzz-EH82, 9zzzz-a0a0g3fe15, 9bact-KY203031, 9bact-t1w006, 9zzzz-a0a0g3fet6, 9bact-KY458164, geoth-g8myf3, 9bact-a0a1s5ql04, 9gamm-a0a1y0ihk7, 9bact-a0a1s5qly6, 9bact-a0a1s5qkg4, 9bact-a0a1s5qkm4, 9gamm-s5tv80, 9gamm-a0a0c4zhg2, 9zzzz-t1b379, 9gamm-KY483646, 9bact-KY458160, 9zzzz-a0a0g3fj57, 9gamm-s5t8349, 9arch-KY203036, 9bact-KY458168, 9zzzz-a0a0g3fes0, 9zzzz-t1be47, 9bact-KY458159, 9zzzz-a0a0g3fh39, 9bact-t1vzd5, 9prot-EH41, 9bact-Lip114, alcbs-q0vt77, 9bact-a0a1s5qke6, 9bact-a0a1s5qkf3, 9prot-SRP030024, 9gamm-s5t532, 9bact-a0a1s5qkl2, 9bact-a0a1s5qkk8, 9zzzz-KY203030, 9zzzz-t1d4I7, 9prot-KY019260, 9bact-a0a1s5qm38, 9arch-KY458161, 9prot-KY010302, 9zzzz-a0a0g3fl25, 9actn-KY010298, 9gamm-s5u059, 9bact-a0a1s5qmi7, 9bact-KY010297, 9bact-KY483642, 9bact-a0a1s5qkj1, 9bact-KY010299, 9bact-KY483648, alcbs-q0vtl7, 9bact-a0a1s5qf1, 9bact-a0a1s5qkg0, 9bact-a0a0h4tgu6, 9bact-MilE3, 9bact-LAE6, 9alte-MGS-MT1, 9bact-r9qzf7, 9gamm-k0c6t6, alcbs-q0vl36, alcbs-q0vlq1, alcbs-q0vq49, bacsu-pnbae, canar-LipB, canan-lipasA, geost-lipas, marav-a1u5n0, pseps-i7k8x5, staep-GEHD, symth-q67mr3, altma-s5cfn7, cycsp-k0c2b8, alcbs-q0vlk5, 9bact-k7qe48, 9bact-MGS-M1, 9bact-MGS-M2, 9bact-a0a0b5kns5, 9zzzz-a0a0g3fej4, 9zzzz-a0a0g3fj60, 9zzzz-a0a0g3fej0, 9zzzz-a0a0g3fj64, 9bact-a0a0b5kc16, 9zzzz-a0a0g3feg6, 9zzzz-a0a0g3feu6 Abstract Esterases receive special attention because their wide distribution in biological systems and environments and their importance for physiology and chemical synthesis. The prediction of esterases substrate promiscuity level from sequence data and the molecular reasons why certain such enzymes are more promiscuous than others, remain to be elucidated. This limits the surveillance of the sequence space for esterases potentially leading to new versatile biocatalysts and new insights into their role in cellular function. Here we performed an extensive analysis of the substrate spectra of 145 phylogenetically and environmentally diverse microbial esterases, when tested with 96 diverse esters. We determined the primary factors shaping their substrate range by analyzing substrate range patterns in combination with structural analysis and protein-ligand simulations. We found a structural parameter that helps ranking (classifying) promiscuity level of esterases from sequence data at 94% accuracy. This parameter, the active site effective volume, exemplifies the topology of the catalytic environment by measuring the active site cavity volume corrected by the relative solvent accessible surface area (SASA) of the catalytic triad. Sequences encoding esterases with active site effective volumes (cavity volume/SASA) above a threshold show greater substrate spectra, which can be further extended in combination with phylogenetic data. This measure provides also a valuable tool for interrogating substrates capable of being converted. This measure, found to be transferred to phosphatases of the haloalkanoic acid dehalogenase superfamily and possibly other enzymatic systems, represents a powerful tool for low-cost bioprospecting for esterases with broad substrate ranges, in large scale sequence datasets. Title: Comparative study of the hydrolytic metabolism of methyl-, ethyl-, propyl-, butyl-, heptyl- and dodecylparaben by microsomes of various rat and human tissues Ozaki H, Sugihara K, Watanabe Y, Fujino C, Uramaru N, Sone T, Ohta S, Kitamura S Ref: Xenobiotica, 43:1064, 2013 : PubMed ESTHER: Ozaki_2013_Xenobiotica_43_1064 PubMedSearch: Ozaki 2013 Xenobiotica 43 1064 PubMedID: 23742084 Inhibitor(s) related to this paper: BNPP Abstract Abstract 1. Hydrolytic metabolism of methyl-, ethyl-, propyl-, butyl-, heptyl- and dodecylparaben by various tissue microsomes and plasma of rats, as well as human liver and small-intestinal microsomes, was investigated and the structure-metabolic activity relationship was examined. 2. Rat liver microsomes showed the highest activity toward parabens, followed by small-intestinal and lung microsomes. Butylparaben was most effectively hydrolyzed by the liver microsomes, which showed relatively low hydrolytic activity towards parabens with shorter and longer alkyl side chains. 3. In contrast, small-intestinal microsomes exhibited relatively higher activity toward longer-side-chain parabens, and showed the highest activity towards heptylparaben. 4. Rat lung and skin microsomes showed liver-type substrate specificity. Kidney and pancreas microsomes and plasma of rats showed small-intestinal-type substrate specificity. 5. Liver and small-intestinal microsomal hydrolase activity was completely inhibited by bis(4-nitrophenyl)phosphate, and could be extracted with Triton X-100. Ces1e and Ces1d isoforms were identified as carboxylesterase isozymes catalyzing paraben hydrolysis by anion exchange column chromatography of Triton X-100 extract from liver microsomes. 6. Ces1e and Ces1d expressed in COS cells exhibited significant hydrolase activities with the same substrate specificity pattern as that of liver microsomes. Small-intestinal carboxylesterase isozymes Ces2a and Ces2c expressed in COS cells showed the same substrate specificity as small-intestinal microsomes, being more active toward longer-alkyl-side-chain parabens. 7. Human liver microsomes showed the highest hydrolytic activity toward methylparaben, while human small-intestinal microsomes showed a broadly similar substrate specificity to rat small-intestinal microsomes. Human CES1 and CES2 isozymes showed the same substrate specificity patterns as human liver and small-intestinal microsomes, respectively. Title: Metabolism of parabens (4-hydroxybenzoic acid esters) by hepatic esterases and UDP-glucuronosyltransferases in man Abbas S, Greige-Gerges H, Karam N, Piet MH, Netter P, Magdalou J Ref: Drug Metab Pharmacokinet, 25:568, 2010 : PubMed ESTHER: Abbas_2010_Drug.Metab.Pharmacokinet_25_568 PubMedSearch: Abbas 2010 Drug.Metab.Pharmacokinet 25 568 PubMedID: 20930423 Abstract Parabens (alkyl esters of 4-hydroxybenzoic acid) are widely used as preservatives in drugs, cosmetic products, and foodstuffs. Safety concerns have recently increased due to the potential health risks associated to exposure to large amounts of these substances. Biotransformation of parabens mainly includes hydrolysis of the ester bond and glucuronidation reactions. The hydrolysis and glucuronidation of a series of six parabens differing by the nature of the alkyl group were investigated in human liver microsomes and plasma, and the major human UDP-glucuronosyltransferase (UGT) isoforms involved in the reaction were identified. Methyl- and ethylparaben were stable in human plasma, with 95% of the initial concentration remaining after 24 h. On the other hand, propyl-, butyl- and benzylparaben concentrations decreased by 50% under similar conditions. In contrast, rapid hydrolysis was measured with human liver microsomes depending on the alkyl chain length, with t(1/2) varying from 22 min for methylparaben to 87 min for butylparaben. All parabens were actively glucuronidated by liver microsomes, in comparison to 4-hydroxybenzoic acid. They were mainly substrates of human recombinant UGT1A1, UGT1A8, UGT1A9, UGT2B7, UGT2B15 and UGT2B17. In conclusion, the parabens were readily metabolized in human liver through esterase hydrolysis and glucuronidation by several UGT isoforms. These results suggest that these parabens do not accumulate in human tissue. Title: Inter-individual variability in esterases in human liver Jewell C, Bennett P, Mutch E, Ackermann C, Williams FM Ref: Biochemical Pharmacology, 74:932, 2007 : PubMed ESTHER: Jewell_2007_Biochem.Pharmacol_74_932 PubMedSearch: Jewell 2007 Biochem.Pharmacol 74 932 PubMedID: 17651701 Abstract Human liver has numerous hydrolytic enzymes involved in metabolism of endogenous and exogenous esters. Of these enzymes, carboxylesterases (EC 3.1.1.1) form an important group which hydrolyses many diverse ester substrates, including pro-ester drugs. Carboxylesterase activity was investigated in liver subcellular fractions from 22 individuals using the general carboxylesterase substrate phenylvalerate and the homologous series of esters methyl-, ethyl-, propyl-, butyl- and benzylparaben. The intra- and inter-individual variation in phenylvalerate and paraben metabolism was compared. Rates of hydrolysis were higher in microsomal fractions than cytosolic fractions for all compounds. The rate of paraben hydrolysis varied depending on the size of the paraben alcohol leaving group, showing a decrease with increasing leaving group size. Comparisons showed that individuals with high rates of hydrolysis towards methyl paraben also showed high rates of hydrolysis to the other parabens and phenylvalerate. Phenylvalerate as a non-specific carboxylesterase substrate was hydrolysed mainly by hCE1 in human livers and there was good correlation with small alcohol leaving group parabens, suggesting hCE1 involvement. Lower correlations with larger alcohol leaving group parabens are consistent with more hCE2 involvement. Title: Purification and characterization of PrbA, a new esterase from Enterobacter cloacae hydrolyzing the esters of 4-hydroxybenzoic acid (parabens) Valkova N, Lepine F, Labrie L, Dupont M, Beaudet R Ref: Journal of Biological Chemistry, 278:12779, 2003 : PubMed ESTHER: Valkova_2003_J.Biol.Chem_278_12779 PubMedSearch: Valkova 2003 J.Biol.Chem 278 12779 PubMedID: 12556461 Gene_locus related to this paper: entcl-PRBA Inhibitor(s) related to this paper: Tosyl-L-lysine-chloromethyl-ketone Abstract The esterase PrbA from Enterobacter cloacae strain EM has previously been shown to confer additional resistance to the esters of 4-hydroxybenzoic acid (parabens) to two species of Enterobacter. The PrbA protein has been purified from E. cloacae strain EM using a three-step protocol resulting in a 60-fold increase in specific activity. The molecular mass of the mature enzyme was determined to be 54,619 +/- 1 Da by mass spectrometry. It is highly active against a series of parabens with alkyl groups ranging from methyl to butyl, with K(m) and V(max) values ranging from 0.45 to 0.88 mM and 0.031 to 0.15 mM/min, respectively. The K(m) and V(max) values for p-nitrophenyl acetate were 3.7 mM and 0.051 mM/min. PrbA hydrolyzed a variety of structurally analogous compounds, with activities larger than 20% relative to propyl paraben for methyl 3-hydroxybenzoate, methyl 4-aminobenzoate, or methyl vanillate. The enzyme showed optimum activity at 31 degrees C and at pH 7.0. PrbA was able to transesterify parabens with alcohols of increasing chain length from methanol to n-butanol, achieving 64% transesterification of 0.5 mm propyl paraben with 5% methanol within 2 h. PrbA was inhibited by 1-chloro-3-tosylamido-4-phenyl-2-butanone and 1-chloro-3-tosylamido-7-amino-2-heptanone (TLCK), with K(i) values of 0.29 and 0.20 mM, respectively, and was irreversibly inhibited by Diisopropyl fluorophosphate (DFP) or diethyl pyrocarbonate. The stoichiometry of addition of DFP to the enzyme was 1:1 and only 1 TLCK molecule was found in TLCK-modified enzyme, as measured by mass spectrometry. Analysis of the tryptic digest of the DFP-modified PrbA demonstrated that the addition of a DFP molecule occurred at Ser-189, indicating the location of the active serine. |
