Enzymatic Recognition Research Articles

Analysis of Substrate Specificity by Lipase for Polyunsaturated Lipids at Different Concentrations

Interfacial activation, specificity and the function of lid domain on microbial lipase, pancreatic lipase, and identical proteins of pancreatic lipase are studied using crystal structure of these proteins. The regioselectivity of lipase for attacking sn-1, sn-2 and sn-3 positions of triacylglycerol are examined and methods for determining fatty acid specificity were studied. In order to investigate the enzymatic recognition in different concentrations to identify how lipase recognizes lipid diversity, substrate specificity by lipase from Rhizomucor miehei for polyunsaturated fatty acyl lipids at 1mM and 100mM was compared. Reaction rates and the specificity constant (apparent Vmax/Km) were measured using native and immobilized lipases. Higher reaction rates for 1mM substrates were noted using immobilized lipases fixed on anion exchange resin with glutaraldehyde and on cation exchange carrier with carbodiimide, indicating that some modification of the basic amino acid in the lid of R. miehei lipase. However, the activation with these bifunctional reagents was not observed for 100mM substrates, indicating that interfacial activation always occurred by the aggregate of 100mM substrate. These characteristics were particularly observed reaction rates in hydrolysis and specificity constants in esterification.

Enzymatic recognition and biological effects of photodynamic damage induced in DNA by 1,6-dioxapyrene plus UVA

The specific recognition of DNA modifications by repair endonucleases was used to characterize DNA damage induced by 1,6-dioxapyrene (1,6-DP) in the presence of ultraviolet light at 365 nm (UVA) in the plasmid YEplac 181. Under cell free conditions, 1,6-DP plus UVA generated lesions are recognized by the UvrABC endonuclease, the proteins Nth, Nfo and Fpg. The number of UvrABC sensitive sites was at least ten-fold higher than that of Fpg or Nth sensitive sites. Moreover, 1,6-DP plus UVA generated single-strand breaks which are the second most frequent lesions. To investigate the biological effect of DNA damage, YEplac181 DNA was treated with 1,6-DP plus UVA and transformed into Escherichia coli or Saccharomyces cerevisae. In Escherichia coli, the transformation efficiency of 1,6-DP plus UVA treated DNA was greatly reduced in the uvrA mutant compared to that in the wild-type strain. However, the transforming efficiency was not affected in Fpg-deficient strains. In Saccharomyces cerevisiae, the transformation efficiency of 1,6-DP plus UVA treated YEplac181 was greatly reduced in the rad14:: URA3 strain. The photobiological effect of 1,6-DP plus UVA was also analysed in haploid yeast strains of various repair capacities. The results show that the yeast strain defective in the nucleotide excision repair pathway ( rad14:: URA3) is hypersensitive to 1,6-DP plus UVA treatment as compared to the parental wild-type strain. It is confirmed that the lethal effect of 1,6-DP plus UVA on wild-type yeast is strongly oxygen dependent, whereas the survival of the rad14:: URA3 mutant only exhibits a minor oxygen dependence. To conclude, our data show that the photodynamic DNA lesions induced by 1,6-DP plus UVA can be recognized and repaired in pro-and eukaryotic cells by the nucleotide excision repair pathway.

Enzymatic Recognition of Acyclic Universal Base Analogues in Oligonucleotides

Abstract The effects of the acyclic analogues 3 and 4 on recognition by commonly used enzymes in recombinant DNA work have been investigated. This report shows that acyclic sugars can function in place of the normal 2′- deoxyribose moiety with some enzymes, expanding the range of possible manipulations.

Mechanism, Specificity and General Properties of the Yeast Enzyme Catalysing the Formation of Inosine 34 in the Anticodon of Transfer RNA

In yeast, inosine is found at the first position of the anticodon (position 34) of seven different isoacceptor tRNA species, while in Escherichia coliit is present only in tRNA Arg. The corresponding tRNA genes all have adenosine at position 34. Using as substrates in vitroT7-runoff transcripts of 31 plasmids carrying each natural of synthetic tRNA gene harbouring an anticodon with adenosine 34, we have characterised a yeast enzyme that catalyses the conversion of adenosine 34 to inosine 34. The homologous E. colienzyme modifies adenosine 34 only in tRNAs with an arginine anticodon ACG. The base conversion occurs by a hydrolytic deamination- type reaction. This was determined by reversed phase high-pressure liquid chromatography/electrospray mass spectrometry analysis of the reaction product after in vitromodification in [ 180]water. This newly characterised tRNA : adenosine 34 deaminase was partially purified from yeast. It has a molecular mass of approximately 75 kDa, and it does not require any cofactor, except magnesium ions, to deaminate adenosine 34 efficiently in tRNA. The observed dependence of the enzymatic reaction on magnesium ions probably reflects the need for a correct tRNA architecture. Enzymatic recognition of tRNA does not depend on the presence of any ‘identity’ nucleoside other than adenosine 34. Likewise, the presence of pseudo-uridine 32 or 1-methyl-guanosine 37 in the anticodon loop does not interfere with inosine 34 biosynthesis. However, the efficacy of adenosine 34 to inosine 34 conversion depends on the nucleotide sequence of the anticodon loop and its proximal stem, the best tRNA substrates being those with a purine at position 35. Mutations that affect the size of the anticodon loop or one of several three-dimensional base-pairs abolish the capacity of the tRNA to be substrate for the yeast tRNA : adenosine 34 deaminase. Evidently, the activity of yeast tRNA : adenosine 34 deaminase depends more on the global structural feature (conformational stability/flexibility) of the L-shaped tRNA substrates than on the identity of any particular nucleotide other than adenosine 34. An apparent K mof 2.3 nM for its natural substrate tRNA Ser(anticodon AGA) was measured. Altogether, these results suggest that a single enzyme can account for the presence of inosine 34 in all seven cytoplasmic A34-containing precursor tRNAs in yeast.

Enzymatic recognition and biological effects of DNA damage induced by 3-carbethoxypsoralen plus UVA

The specific recognition of DNA modifications by repair endonucleases was used to characterize damage induced by 3-carbethoxypsoralen (3-CPs) plus UvA in M13mp8 replicative form I (RF-I) DNA. Under the conditions used, 3-CPs plus UVA generates DNA base modifications which are recognized by the UvrABC complex and the Fpg protein of E. coli. The rate of formation of UvrABC sensitive sites is 3–4-fold higher than that of Fpg sensitive sites. In addition a small number of sites of base loss (sensitive to Nfo protein) were observed. M13mp8 RF-I DNA treated with 3-CPs plus UVA was tested for transfection efficiency in E. coli mutants defective in either Fpg protein and/or UvrABC complex. The survival of 3-CPs plus UVA damaged M13mp8 RF-I DNA was significantly reduced when transfected into uvrA mutants compared to that in the wild-type strain. On the other hand, the survival of 3-CPs plus UVA damaged RF-I DNA was not altered in fpg-1 mutants. These results show that nucleotide excision repair mediated by the UvrABC complex is the major repair pathway involved in the elimination of lethal lesions induced in DNA by 3-CPs plus UVA. Our data suggest that in vitro exposure of M13mp8 RF-I DNA to 3-CPs plus UVA produces predominantly thymine photoaddition and to a lesser extent guanine photooxidation partially due to singlet oxygen generated during photoreaction. The photoaddition products are primarly responsible for the observed lethal effect.

Efficient optical resolution of 2-(4-chlorophenoxy)propanoic acid with lipase by the use of organosilicon compounds as substrate: the role of silicon atom in enzymatic recognition

Comparative studies were made of the use as acyl acceptor of organosilicon compounds [Me 3Si(CH 2) nOH] and the corresponding carbon compounds [Me 3C(CH 2) nOH] in the stereoselective esterification of 2-(4-chlorophenoxy)propanoic acid by the use of lipase OF 360 of Candida cylindracea in water-saturated benzene. The organosilicon compounds were effectively used as the substrates for the esterification of the d-acid enantiomer. Of the organosilicon compounds of different chain-length between the silicon atom and the hydroxyl group, trimethylsilylmethanol ( n = 1) enabled the esterification reaction to be both fast and highly stereoselective, which was difficult with conventional substrates such as its carbon counterpart. On the other hand, no difference was observed between trimethylsilylethanol ( n = 2) and its carbon analogue (3,3-dimethylbutanol) in the enzymatic activity and the stereoselectivity. These results indicate that the silicon atom behaved as a mimic carbon atom for lipase in the case of trimethylsilylethanol but was effective in enhancing the reactivity of trimethylsilylmethanol. The difference could be explained on the basis of the properties of silicon atom, such as its low electronegativity and big atomic radius compared with the carbon atom.

Enzymatic recognition of diastereomeric esters

Aiming to improve the enantioselectivity of enzymatic resolution of esters, lipase catalyzed hydrolysis of (D)- and (L-2-chloropropanoates of four racemic alcohols and transesterification of ethyl (DL)-2-chloropropanoate with optically pure alcohols was investigated. Thus, ( rac)- endo-2-norbornyl (L)-2-chloropropanoate was hydrolyzed by lipase P about 5 times more selectively than its corresponding (D)-counterpart and optically pure (1 R2 S,5 R)-menthol was obtained by transesterification of its racemate with ethyl (D)-2-chloropropanoate using Candida cylindracea (CC) lipase. From the results obtained it seems obvious that lipases CC and P mainly can recognize the chirality of an alcohol moiety rather than that of an acid.

Enzymatic recognition of DNA modifications induced by singlet oxygen and photosensitizers

DNA modifications induced either by photosensitization (illumination in the presence of methylene blue) or by chemically generated singlet oxygen (thermal decomposition of an 1,4-etheno-2,3-benzodioxin) are recognized and incised by repair endonucleases present in crude bacterial cell extracts. Only a small fraction of the incised modifications are sites of base loss (AP-sites) sensitive to exonuclease III, endonuclease IV from E. coli or to the UV-endonuclease from M. luteus. Cell extracts from E. coli strains overproducing or defective in endonuclease III recognize the modifications induced by illumination in the presence of methylene blue just as well as do those from wild-type E. coli strains. This indicates that dihydropyrimidine derivatives, which are characteristic of hydroxyl radical-induced DNA modifications, are absent. In contrast, most of the modifications induced are not recognized by a cell extract from a fpg strain defective in formamidopyrimidine-DNA glycosylase FPG protein). Furthermore, incision by a cell extract from an E. coli strain overproducing FPG protein takes place at much lower protein concentration than with the wild-type strain. Experiments with purified FPG protein confirm that this enzyme is responsible for the recognition of singlet oxygen-induced DNA base modifications.

Calcium affects the spontaneous degradation of aspartyl/asparaginyl residues in calmodulin

We have previously shown that the D-aspartyl/L-isoaspartyl protein carboxyl methyltransferase recognizes two major sites in affinity-purified preparations of bovine brain calmodulin that arise from spontaneous degradation reactions. These sites are derived from aspartyl residues at positions 2 and 78, which are located in apparently flexible regions of calmodulin. We postulated that this flexibility was an important factor in the nonenzymatic formation and enzymatic recognition of D-aspartyl and/or L-isoaspartyl residues. Because removal of Ca2+ ions from this protein may also lead to increased flexibility in the four Ca2+ binding regions, we have now characterized the sites of methylation that occur when calmodulin is incubated in buffers with or without the calcium chelator ethylene glycol bis(beta-aminoethyl ether)-N,N,-N',N'-tetraacetic acid (EGTA). Calmodulin was treated at pH 7.4 for 13 days at 37 degrees C under these conditions and was then methylated with erythrocyte D-aspartyl/L-isoaspartyl methyltransferase isozyme I and S-adenosyl-L-[methyl-3H]methionine. The 3H-methylated calmodulin product was purified by reverse-phase HPLC and digested with various proteases including trypsin, chymotrypsin, endoproteinase Lys-C, clostripain, and Staphylococcus aureus V8 protease, and the resulting peptides were separated by reverse-phase HPLC. Peptides containing Asp-2 and Asp-78, as well as calcium binding sites II, III, and IV, were found to be associated with radiolabel under these conditions. When calmodulin was incubated under the same conditions in the presence of calcium, methylation at residues in the Ca2+ binding regions was not observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetyl-coenzyme A:polysialic acid O-acetyltransferase from K1-positive Escherichia coli. The enzyme responsible for the O-acetyl plus phenotype and for O-acetyl form variation.

The capsular polysaccharide of Escherichia coli K1 is a linear polymer of N-acetylneuraminic acid in alpha-2,8 linkage. Certain substrains of E. coli K1 (designated OAc+) modify the polysaccharide by O-acetylation of the sialic acids. We demonstrate here an acetyl-coenzyme A: polysialosyl O-acetyltransferase activity that is found only in E. coli K1 OAc+ substrains. When form variation between the O-acetyl-positive and -negative states occurred in strain D698:K1, the fluctuations were accompanied by appropriate changes in the expression of enzyme activity. Thus, expression of this enzyme can account for the OAc+ phenotype and for the form variation between OAc+ and OAc-. The enzyme was solubilized in nonionic detergent and freed of endogenous acceptor activity by DEAE-cellulose chromatography, and its general properties were determined. Analysis of the reaction product showed a highly preferential acetylation reaction that was confined to polysialosyl units of greater than 14 residues. Acetyl groups were shown to be transferred to both the 7- and the 9-positions of the sialic acid residues. The partially purified enzyme was stable even after prolonged incubation at 57 degrees C. In contrast, any further purification resulted in loss of activity, even at 4 degrees C. Treatment of the stable enzyme with a polysialic acid-specific endoneuraminidase caused a similar loss of enzyme stability. This effect of the endoneuraminidase could be protected against by the addition of exogenous polysialic acid. This indicates that the partially purified enzyme contains traces of endogenous polysialic acid substrate that are required for the stability of the enzyme. Finally, the enzyme can O-acetylate the polysialic acid chains on the eucaryotic protein neural cell adhesion molecule, suggesting that enzymatic recognition of the substrate requires only the polysialic acid sequence.

Monoterpene cyclases: Physicochemical features required for pyrophosphate binding determined from inhibition by structural analogs

Monoterpene cyclases catalyze the divalent metal ion-dependent conversion of geranyl pyrophosphate, the ubiquitous C 10 intermediate of isoprenoid biosynthesis, to a variety of monoterpene skeletons, and the pyrophosphoryl moiety is a primary determinant for substrate binding by these enzymes. To determine what specific features of this functional group are critical for enzymatic recognition, inorganic pyrophosphate and a series of structurally related analogs were examined as inhibitors of geranyl pyrophosphate: (+)-α-pinene cyclase and geranyl pyrophosphate:(+)-bornyl pyrophosphate cyclase from sage ( Salvia officinalis). Analysis of trends in the magnitude of inhibition by the analogs relative to inorganic pyrophosphate indicated that the combination of ionization state (formal charge) at the enzymatic pH optimum, ability to chelate divalent metal ions, and intramolecular flexibility is required for effective interaction with both cyclases. Only when all of these criteria are met is inhibition of cyclization comparable to that observed with inorganic pyrophosphate.

Structure of an adenine-cytosine base pair in DNA and its implications for mismatch repair.

Mutational pathways rely on introducing changes in the DNA double helix. This may be achieved by the incorporation of a noncomplementary base on replication or during genetic recombination, leading to substitution mutation. In vivo studies have shown that most combinations of base-pair mismatches can be accommodated in the DNA double helix, albeit with varying efficiencies. Fidelity of replication requires the recognition and excision of mismatched bases by proofreading enzymes and post-replicative mismatch repair systems. Rates of excision vary with the type of mismatch and there is some evidence that these are influenced by the nature of the neighbouring sequences. However, there is little experimental information about the molecular structure of mismatches and their effect on the DNA double helix. We have recently determined the crystal structures of several DNA fragments with guanine X thymine and adenine X guanine mismatches in a full turn of a B-DNA helix and now report the nature of the base pairing between adenine and cytosine in an isomorphous fragment. The base pair found in the present study is novel and we believe has not previously been demonstrated. Our results suggest that the enzymatic recognition of mismatches is likely to occur at the level of the base pairs and that the efficiency of repair can be correlated with structural features.

In vitro enzymatic recognition of DNA modified by O,O'-diacetyl or O-acetyl derivatives of the carcinogen 4-hydroxyaminoquinoline-1-oxide.

Purified DNA was modified in vitro by 3H-labelled O-acetyl or O,O'-diacetyl-4-hydroxyaminoquinoline-1-oxide (Ac4HAQO or di Ac-4HAQO). It was then subjected to the action of the single-stranded DNA specific nuclease S1 and the digested fractions were analysed. For both types of modified DNA, the release of non-modified nucleotides was faster than the release of modified nucleotides. This result is at variance with that obtained with acetoxy-acetylaminofluorene-modified DNA: in the latter case, the modified nucleotides were preferentially released. The results suggest that the S1 endonuclease can recognize different conformational changes in DNA, which depend on the carcinogen used. The enzymatic activity (or activities) present in Micrococcus luteus cell extracts released ethanol-soluble products from Ac-4HAQO modified DNA.

Enzymatic deacetylation of a synthetic peptide fragment of histone H4

FEBS LettersVolume 106, Issue 1 p. 93-96 Full-length articleFree Access Enzymatic deacetylation of a synthetic peptide fragment of histone H4 Aline Kervabon, Aline Kervabon Equipe de Recherche de Biophysique no. 140, CNRS, USTL, 34060 Montpellier, FranceSearch for more papers by this authorJean Mery, Jean Mery Equipe de Recherche de Biophysique no. 140, CNRS, USTL, 34060 Montpellier, FranceSearch for more papers by this authorJoseph Parello, Joseph Parello Equipe de Recherche de Biophysique no. 140, CNRS, USTL, 34060 Montpellier, FranceSearch for more papers by this author Aline Kervabon, Aline Kervabon Equipe de Recherche de Biophysique no. 140, CNRS, USTL, 34060 Montpellier, FranceSearch for more papers by this authorJean Mery, Jean Mery Equipe de Recherche de Biophysique no. 140, CNRS, USTL, 34060 Montpellier, FranceSearch for more papers by this authorJoseph Parello, Joseph Parello Equipe de Recherche de Biophysique no. 140, CNRS, USTL, 34060 Montpellier, FranceSearch for more papers by this author First published: October 01, 1979 https://doi.org/10.1016/0014-5793(79)80702-4Citations: 17AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume106, Issue1October 01, 1979Pages 93-96 ReferencesRelatedInformation

Chemical studies on histone acetylation using a synthetic peptide fragment of histone H4

FEBS LettersVolume 98, Issue 1 p. 152-156 Full-length articleFree Access Chemical studies on histone acetylation using a synthetic peptide fragment of histone H4 Aline Kervabon, Aline Kervabon Equipe de Recherche de Biophysique No 140, CNRS, USTL, 34060 Montpellier, FranceSearch for more papers by this authorJoseph Parello, Joseph Parello Equipe de Recherche de Biophysique No 140, CNRS, USTL, 34060 Montpellier, FranceSearch for more papers by this authorJean Mery, Jean Mery Equipe de Recherche de Biophysique No 140, CNRS, USTL, 34060 Montpellier, FranceSearch for more papers by this author Aline Kervabon, Aline Kervabon Equipe de Recherche de Biophysique No 140, CNRS, USTL, 34060 Montpellier, FranceSearch for more papers by this authorJoseph Parello, Joseph Parello Equipe de Recherche de Biophysique No 140, CNRS, USTL, 34060 Montpellier, FranceSearch for more papers by this authorJean Mery, Jean Mery Equipe de Recherche de Biophysique No 140, CNRS, USTL, 34060 Montpellier, FranceSearch for more papers by this author First published: February 01, 1979 https://doi.org/10.1016/0014-5793(79)80172-6Citations: 8AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References 1 A. Ruiz-Carrillo, L.J. Wangh, V.G. Allfrey, Science, 190, (1975), 117– 128. 2 G.H. Dixon, E.P.M. Candido, B.M. Honda, A.J. Louie, A.R. Mac Leod, M.T. Sung, D.W. Fitzsimons G.E.W. Wolstenholme The structure and function of chromatin (1975), Elsevier Amsterdam 229– 258. Ciba Found. Symp. 28 (new ser.) 3 E.P.M. Candido, G.H. Dixon, J. Biol. Chem., 247, (1972), 5506– 5510. 4 I. Sures, D. Gallwitz, FEBS Lett., 53, (1975), 92– 94. 5 M. Gourévitch, P. Puigdomènech, A. Cavé, G. Etienne, J. Méry, J. Parello, Biochimie, 56, (1974), 967– 985. 6 E.W. Johns, Biochem. J., 92, (1964), 55– 59. 7 S. Panyim, R. Chalkley, Arch. Biochem. Biophys., 130, (1969), 337– 346. 8 P.G. Pietta, G.R. Marshall, Chem. Commun., (1970), 650– 651. 9 J. Méry, Thèse 3ème cycle (1977), USTL Montpellier, France 10 D.R. Hewish, L.A. Burgoyne, Biochem. Biophys. Res. Commun., 52, (1973), 504– 510. 11 D. Gallwitz, Biochem. Biophys. Res. Commun., 40, (1970), 236– 242. 12 G.A. Bray, Anal. Biochem., 1, (1960), 279– 285. 13 J.F. Riordan, B.L. Vallee, Adv. in Enzymology, 25, (1972), 494– 499. 14 B.M. Honda, G.H. Dixon, E.P.M. Candido, J. Biol. Chem., 250, (1975), 8681– 8685. 15 B.M. Honda, E.P.M. Candido, G.H. Dixon, J. Biol. Chem., 250, (1975), 8686– 8689. 16 D. Gallwitz, I. Sures, Biochim. Biophys. Acta, 263, (1972), 315– 328. 17 D. Gallwitz, E.K.F. Bautz Regulation of transcription and translation in eukaryotes (1973), Springer-Verlag Berlin 37– 57. Citing Literature Volume98, Issue1February 01, 1979Pages 152-156 ReferencesRelatedInformation

Determination of the intracellular concentration of 5-phosphoribosyl-1-pyrophosphate in cultured mammalian fibroblasts

The properties of an assay for the 5-phosphoribosyl-1-pyrophosphate (PRPP) content of cultured mammalian fibroblasts are described. The assay is based upon the PRPP-dependent release of 14CO 2 from [ carboxyl- 14C]orotic acid by a commercially available preparation of yeast orotidine-5′-monophosphate pyrophosphorylase and orotidine-5′-monophosphate decarboxylase. The advantages of the assay include the fact that it is based on the enzymatic recognition of PRPP, employs an irreversible reaction, and does not involve either the chromatographic separation of substrate and product or the purification of a phosphoribosyltransferase. The disadvantage of the assay is that the efficiency of PRPP measurement varies somewhat, in part because the yeast enzyme preparation contains 5′-nucleotidase activity. A calibration procedure is described which corrects for variation in efficiency both between and within experiments. This procedure seems to yield highly reliable estimates of PRPP content. The assay will readily detect 0.6 nmol, and the cell strain studied contained 7.76 ± 1.14 nmol of PRPP 10 7 cells .