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PubMed1996

Substrate specificity and mode of action of acetylxylan esterase from Streptomyces lividans.

The substrate specificity of purified acetylxylan esterase (AcXE) from Streptomyces lividans was investigated on partially and fully acetylated methyl glycopyranosides. The enzyme exhibited deacetylation regioselectivity on model compounds which provided insights pertaining to its function in acetylxylan degradation. The enzyme catalyzed double deacetylation of methyl 2,3,4-tri-O-acetyl-beta-D-xylopyranoside and methyl 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside at positions 2 and 3. Two methyl xylopyranoside diacetates, which had a free hydroxyl group at position 2 or 3, i.e. the derivatives that most closely mimic monoacetylated xylopyranosyl residues in acetylxylan, were deacetylated 1 to 2 orders of magnitude faster than methyl 2,3,4-tri-O-acetyl-beta-D-xylopyranoside and methyl 2,3-di-O-acetyl-beta-D-xylopyranoside. These observations explain the double deacetylation. The second acetyl group is released immediately after the first one is removed from the fully acetylated methyl beta-D-xylo- and -glucopyranoside. The results suggest that in acetylxylan degradation the enzyme rapidly deacetylates monoacetylated xylopyranosyl residues, but attacks doubly acetylated residues much more slowly. Evidence is also presented that the St. lividans enzyme could be the first real substrate-specific AcXE.

Acetylation↗
PubMed2006

The three desulfoglucosinolate sulfotransferase proteins in Arabidopsis have different substrate specificities and are differentially expressed.

Sulfotransferases (SOTs) catalyse the transfer of a sulfate group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to an appropriate hydroxy group of various substrates with the parallel formation of 3'-phosphoadenosine 5'-phosphate. In Arabidopsis thaliana, 18 SOT proteins (AtSOT) have been identified. Three of them, AtSOT16, AtSOT17 and AtSOT18, catalyse the sulfation of desulfoglucosinolates. The proteins were expressed in Escherichia coli, purified by affinity chromatography and used for enzyme kinetic studies. By establishing two types of enzyme assay using both 35S-labelled and unlabelled PAPS, separation of the products by HPLC, and detection of the products by monitoring radioactivity or UV absorption, the substrate specificities of the three AtSOT proteins were determined. They show different maximum velocities with several desulfoglucosinolates as substrates and differ in their affinity for desulfobenzylglucosinolate and PAPS. The sequences encoding AtSOT18 were amplified from Arabidopsis ecotypes C24 and Col0; the two expressed proteins differ in two out of 350 amino acids. These amino-acid variations led to different substrate specificities. Exchange of one of the two amino acids in AtSOT18 from C24 to the respective amino acid in AtSOT18 from Col0 gave the C24 protein the same substrate specificity as the wild-type AtSOT18 protein from Col0. All three desulfoglucosinolate AtSOT proteins are localized in the cytoplasm, as demonstrated by transient expression of fusion constructs with the green fluorescent protein in Arabidopsis protoplasts. Northern blot analysis indicated differential expression of the three AtSOT genes in plant organs and tissues at different developmental stages and during a light/darkness cycle. High (500 microM) and low (50 microM) sulfate concentrations in the medium did not influence the levels of expression.

Arabidopsis↗
PubMedDate not supplied

[RNAse substrate specificity in Acholeplasma laidlawii PG-8].

A substrate specificity of RNAses of A. laidlawii PG-8 to polynucleotides - poly (C), poly (U), poly (A) has been studied. Due to the data obtained both intracellular and extracellular RNAses of A. laidlawii possess similar specificity to different polynucleotides. Both RNAses preferentially break cytidine-bonds. Specificity of the studied enzymes in respect to polyuridylic and polyadenylic acids was less expressed.

Acholeplasma laidlawii↗
PubMed1988

On the substrate specificity of rat liver phospholipase A1.

The substrate specificity of purified phospholipase A1 was studied using mixed micelles of phospholipid and Triton X-100. The kinetic analysis employed determined Vmax, Ks (a dissociation constant for the phospholipase A1-mixed micelle complex), and Km (the Michaelis constant for the catalytic step which reflects the binding of the enzyme to the substrate in the interface). The order of Vmax values was phosphatidic acid greater than phosphatidylethanolamine greater than phosphatidylcholine greater than phosphatidylserine. The order of Ks values was phosphatidylcholine greater than phosphatidylethanolamine greater than phosphatidic acid greater than phosphatidylserine; the order of Km values was phosphatidic acid greater than phosphatidylethanolamine = phosphatidylserine greater than phosphatidylcholine. When present together, phosphatidylcholine inhibited the hydrolysis of phosphatidylethanolamine but phosphatidylethanolamine did not affect the hydrolysis of phosphatidylcholine. Sphingomyelin, phosphatidylcholine plasmalogen, and phosphatidylethanolamine plasmalogen had no effect on the hydrolysis of phosphatidylethanolamine. The effects of the reaction products, lysolipids and/or fatty acids, were also considered for their influence on phosphatidylethanolamine hydrolysis catalyzed by phospholipase A1. Free fatty acid was found to inhibit, whereas lysophospholipids stimulated hydrolysis of phosphatidylethanolamine. In a mixture of 1,2- and 1,3-diacylglycerides in mixed micelles, only the acyl chain at the sn-1 position of the 1,2 compound was hydrolyzed. Surface charge did not modulate the hydrolysis of phosphatidylcholine vesicles or mixed micelles. In conclusion, it is hypothesized that steric hindrance at position 3 of the glycerol regulates substrate binding in the active site and that an acyl group in position 1 is favored over a vinyl ether linkage for binding.

Animals↗
PubMed1997

Studies on the substrate specificity of Ca2+/calmodulin-dependent protein kinase kinase alpha.

Ca2+/calmodulin-dependent protein kinase (CaM-kinase) kinase a, which is known to activate CaM-kinases IV and I by phosphorylation of Thr196 and Thr177, respectively, can only phosphorylate Thr196 among many phosphorylation sites of CaM-kinase IV [Kitani, T., Okuno, S., and Fujisawa, H. (1997) J. Biochem. 121, 804-810], indicating its high degree of substrate specificity. In the present study, the substrate specificity of CaM-kinase kinase a was examined using various proteins and synthetic peptides as substrates as a means to address its physiological function. Among a number of proteins and synthetic peptides, including several known as good substrates for various protein kinases, only CaM-kinases IV and I and peptides containing the sequence surrounding Thr196 of CaM-kinase IV or Thr177 of CaM-kinase I were significantly phosphorylated by CaM-kinase kinase alpha, while the heat-denatured (at 60 degrees C for 5 min) CaM-kinases IV and I were not phosphorylated. Peptides containing the phosphorylation site of CaM-kinase IV or I were far less active as substrates for CaM-kinase kinase a than were native CaM-kinase IV or I. Thus, CaM-kinase kinase a showed a high degree of substrate specificity, recognizing not only specific amino acid sequences but also the native conformation of CaM-kinases IV and I.

Adenosine Triphosphate↗
PubMed1989

Selection of amidases with novel substrate specificities from penicillin amidase of Escherichia coli.

To obtain amidases with novel substrate specificity, the cloned gene for penicillin amidase of Escherichia coli ATCC 11105 was mutagenized and mutants were selected for the ability to hydrolyze glutaryl-(L)-leucine and provide leucine to Leu- host cells. Cells with the wild-type enzyme did not grow in minimal medium containing glutaryl-(L)-leucine as a sole source of leucine. The growth rates of Leu- cells that expressed these mutant amidases increased as the glutaryl-(L)-leucine concentration increased or as the medium pH decreased. Growth of the mutant strains was restricted by modulation of medium pH and glutaryl-(L)-leucine concentration, and successive generations of mutants that more efficiently hydrolyzed glutaryl-(L)-leucine were isolated. The kinetics of glutaryl-(L)-leucine hydrolysis by purified amidases from two mutants and the respective parental strains were determined. Glutaryl-(L)-leucine hydrolysis by the purified mutant amidases occurred most rapidly between pH 5 and 6, whereas hydrolysis by wild-type penicillin amidase at this pH was negligible. The second-order rate constants for glutaryl-(L)-leucine hydrolysis by two "second-generation" mutant amidases, 48 and 77 M-1 s-1, were higher than the rates of hydrolysis by the respective parental amidases. The increased rates of glutaryl-(L)-leucine hydrolysis resulted from both increases in the molecular rate constants and decreases in apparent Km values. The results show that it is possible to deliberately modify the substrate specificity of penicillin amidase and successively select mutants with amidases that are progressively more efficient at hydrolyzing glutaryl-(L)-leucine.

Amidohydrolases↗
PubMed1994

Replacement of serine 237 in class A beta-lactamase of Proteus vulgaris modifies its unique substrate specificity.

The chromosomal beta-lactamase gene of Proteus vulgaris K1 was cloned and sequenced. The gene comprises 813 nucleotides and codes for the mature enzyme of 29,655 Da, comprising 271 amino acids. The K1 beta-lactamase showed 30-70% similarity, in the overall amino acid sequence, to class A beta-lactamases of Gram-negative bacteria. However, the K1 beta-lactamase differs from most class A enzymes in having a unique substrate specificity as a cephalosporinase, its spectrum extending to even oxyiminocephalosporins. To clarify the relationship between its unique substrate specificity and specific amino acid residues, alignment of the amino acid sequence of the K1 beta-lactamase with those of class A beta-lactamases was performed, and Ala104 and Ser237 were found to be candidates. Ala104 and Ser237 were replaced with glutamic acid and alanine, respectively, which are commonly found in other class A beta-lactamases. The substitution at position 104 had no effect on the enzyme activity or the substrate specificity. The amino acid replacement at position 237, however, reduced the kcat/Km value for an oxyiminocephalosporin (cefuroxime) to 17% of that in the case of the wild-type enzyme, whereas the mutant enzyme showed a higher kcat/Km value for benzylpenicillin, 3 times, than that of the wild-type enzyme. These results indicated that Ser237 is one of the residues responsible for the unique substrate specificity of the P. vulgaris beta-lactamase.

Alanine↗
PubMed1995

In situ substrate specificity and ultrastructural localization of polyamine oxidase activity in unfixed rat tissues.

Data concerning the substrate specificity and the exact intracellular localization of the polyamine-catabolizing enzyme polyamine oxidase are conflicting. Biochemical studies have shown that N1-acetylation of spermine and spermidine dramatically increases the specificity of these compounds for peroxisomal polyamine oxidase to produce spermidine and putrescine, respectively. On the other hand, polyamine oxidase activity was demonstrated histochemically both in peroxisomes and in cytoplasm of several tissues, using spermidine and/or spermine as substrate. To elucidate the in situ substrate specificity of polyamine oxidase and the localization of its activity, enzyme activity was detected in rat liver, kidney, and duodenum at the light and electron microscopic levels. For this purpose, unfixed cryostat sections were applied to avoid changes in enzyme activity owing to chemical fixation. Spermine, spermidine, their N1-acetylated forms, and putrescine were used as substrates, and cerium ions as capturing agent for H2O2. Control reactions were performed in the absence of substrate or in the presence of substrate and specific oxidase inhibitors. At the light microscopic level, final reaction product specifically generated by polyamine oxidase activity was found exclusively in a granular form in hepatocytes, epithelial cells of proximal tubules of the kidney, and epithelial cells of duodenal villi with N1-acetylspermidine or N1-acetylspermine as substrates. Final reaction product was not observed in any of the tissues after incubation in the presence of putrescine, spermidine, or spermine. Formation of specific final reaction product was prevented by incubation in the presence of a specific polyamine oxidase inhibitor, but it was not affected by a diamine oxidase inhibitor. Ultrastructural studies revealed that polyamine oxidase activity is localized exclusively to the matrix of peroxisomes of kidney and liver and to microperoxisomes of the duodenum. The localization patterns obtained with unfixed tissues are in agreement with biochemical data. Strong intraperoxisomal, interperoxisomal, and intercellular heterogeneity in polyamine oxidase activity was found in all tissues investigated.

Animals↗
PubMed1987

Insight into the active site of Streptomyces cystathionine gamma-lyase based on the results of studies on its substrate specificity.

The results of studies on the substrate specificities of elimination and replacement reactions allowed insight into the active and regulatory sites of Streptomyces phaeochromogenes cystathionine gamma-lyase (L-cystathionine cysteine-lyase (deaminating), EC 4.4.1.1). The enzyme has an active site and a regulatory site. The active site consists of two subsites; one recognizes the L-forms of amino acids (L-homoserine and L-moieties of cystathionine isomers) and the other shows affinity for thiol compounds with a carboxyl group. The regulatory site is specific for L-cysteine and has no affinity for ordinary thiol compounds, such as 3-mercaptopropionate and thioglycolate.

Allosteric Site↗
PubMed1981

Studies on T4-head maturation. 2. Substrate specificity of gene-49-controlled endonuclease.

The substrate specificity of 49+-enzyme was investigated in vitro. The enzyme showed a marked preference for rapidly sedimenting T4 DNA (greater than 1000 S) when helix-destabilizing proteins from Escherichia coli or phage T4 were added to the reaction. Regular replicative T4 DNA (200-S DNA) or denatured T4 DNA was not cleaved by the enzyme in the presence of these proteins but if they were omitted from the reaction both DNAs become good substrates for the enzyme. 200-S DNA was cleaved at its natural sites of single strandedness which occur at one-genome intervals. Gaps in T4 DNA which were constructed by treatment of a nicked DNA with exonuclease III were also cleaved by 49+-enzyme in the absence of helix-destabilizing proteins. Single-stranded T4 DNA was extensively degraded and up to 50% of the material was found to be acid-soluble in a limit digest. The degradation products were predominantly oligonucleotides of random size. No preference for a 5'-terminal nucleotide was observed in material from a limit digest with M13 DNA. Double-stranded DNA was nicked upon exposure to 49+-enzyme and double-strand breakage finally occurred by an accumulation of single-strand interruptions. No acid-soluble material was produced from native T4 DNA. The introduction of nicks in native DNA did not improve its properties as a substrate for the enzyme. Double-stranded DNA was about 100-fold less sensitive to the enzyme than single-stranded DNA.

DNA, Viral↗
PubMed1995

Substrate specificity of the lipase from Candida parapsilosis.

Substrate specificity of the acyltransferase activity of the lipase (EC 3.1.1.3) from Candida parapsilosis CBS 604 was studied in aqueous media. The specificity toward both acid and alcohol parts of a large number of acylglycerols and aliphatic esters was investigated. This lipase showed a high activity in the presence of esters with long-chain fatty acids and particularly unsaturated fatty acids with a cis-delta 9 double bond. It was observed that the activity profile depended not only on the alcohol part of the acyl ester, but also on the temperature of the reactant medium. The best lipid substrates had their melting point between -40 to +20 degrees C, 14 to 18 carbon atoms in the acyl group and 1 to 4 carbon atoms in the alkyl group. The enzyme, defined as an acyltransferase in a previous paper, showed a high affinity for primary and secondary alcohols with a short carbon chain (1 to 5 carbon atoms) as acyl acceptors. The influence of free alcohols in the reactant medium on the hydrolysis and alcoholysis activities of the enzyme is discussed. Two phenomena seem to be involved, depending on the alcohol: competition with water for the acyltransfer reaction and lipid substrate dilution when the alcohol places at the oil/water interface.

Alcohols↗
PubMed1988

Substrate specificity of Staphylococcus aureus (TEN5) lipases with isomeric oleoyl-sn-glycerol ethers as substrates.

For the first time fully protected substrates with only one hydrolyzable ester bond have been used to analyze the substrate specificity of microbial lipases. In these substrates the ester is attached to the glycerol molecule in a precisely defined position. The use of three different substituents generates chirality and thus allows the analysis of positional specificities of individual lipases. Therefore, these new substrates have been used to study the enzymatic activities of two closely related lipases isolated from Staphylococcus aureus (TEN5) designated the 44 and 43 kDa lipase. The lipases, especially the 44 kDa molecule, show a high specificity for the hydrolysis of the ester in the sn-1 position (S-configuration), which is hydrolyzed by a factor of ten faster than that in the sn-3 position. In addition, the study demonstrates for the first time that the rate of hydrolysis of a fatty acid ester attached to the sn-2 position of glycerol by microbial lipases depends on the configuration of the substrate molecule.

Glyceryl Ethers↗
PubMed2003

Beta-glycosylamidine as a ligand for affinity chromatography tailored to the glycon substrate specificity of beta-glycosidases.

An affinity adsorbent for beta-glycosidases has been prepared by using beta-glycosylamidine as a ligand. beta-Glucosylamidine and beta-galactosylamidine, highly potent and selective inhibitors of beta-glucosidases and beta-galactosidases, respectively, were immobilized by a novel one-pot procedure involving the addition of a beta-glycosylamine and 2-iminothiolane.HCl simultaneously to a matrix modified with maleimido groups via an appropriate spacer to give an affinity adsorbent for beta-glucosidases and beta-galactosidases, respectively. This one-pot procedure enables various beta-glycosylamidine ligands to be formed and immobilized conveniently according to the glycon substrate specificities of the enzymes. A crude enzyme extract from tea leaves (Camellia sinensis) and a beta-galactosidase from Penicillium multicolor were chromatographed directly on each affinity adsorbent to give a beta-glucosidase and a beta-galactosidase to apparent homogeneity in one step by eluting the column with glucose or by a gradient NaCl elution, respectively. The beta-glucosidase and beta-galactosidase were inhibited competitively by a soluble form of the corresponding beta-glycosylamidine ligand with an inhibition constant (K(i)) of 2.1 and 0.80 microM, respectively. Neither enzyme was bound to the adsorbent with a mismatched ligand, indicating that the binding of the glycosidases was of specific nature that corresponds to the glycon substrate specificity of the enzymes. The ease of preparation and the selective nature of the affinity adsorbent should promise a large-scale preparation of the affinity adsorbent for the purification and removal of specific glycosidases according to their glycon substrate specificities.

Amidines↗
PubMed1992

Orotidylate decarboxylase: insights into the catalytic mechanism from substrate specificity studies.

Pyrimidine nucleotides were tested as substrates for pure yeast orotidylate decarboxylase in an attempt to gain insight into the nature of the catalytic mechanism of the enzyme. Substitutions of the 5-position in the pyrimidine ring of the orotidylate substrate resulted in compounds that are either excellent inhibitors or substrates of the enzyme. The 5-bromo- and 5-chloroorotidylates are potent inhibitors while the 5-fluoro derivative is a good substrate with a turnover number 30 times that observed with orotidylate. When carbon 5 of the pyrimidine ring is replaced by nitrogen in 5-azaorotidylate, the resulting compound is unstable in solution with a half-life of 25 min at pH 6. However, studies with freshly generated 5-azaorotidylate show that an enzyme-dependent reaction occurs, presumably decarboxylation. This enzyme reaction follows simple Michaelis-Menten kinetics. Because the 5-aza group is not electrophilic, an enzyme mechanism utilizing a nucleophilic addition of the enzyme at the 5-position is ruled out. We also present studies that are not compatible with a mechanism requiring the formation of a Schiff's base prior to decarboxylation. The enzyme is tolerant of modest substitution at the 4-position, for the 4-keto group can be replaced with a thioketone. However, no catalysis is observed when the same substitution is made at the 2-position. Similarities in the substrate specificity of orotate phosphoribosyltransferase and orotidylate decarboxylase led us to compare the amino acid sequences of the two enzymes; significant (20%) sequence homology was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1986

Substrate specificity of a mammalian DNA repair endonuclease that recognizes oxidative base damage.

The substrate specificity of a calf thymus endonuclease on DNA damaged by UV ligh, ionizing radiation, and oxidizing agents was investigated. End-labeled DNA fragments of defined sequence were used as substrates, and the enzyme-generated scission products were analyzed by using DNA sequencing methodologies. The enzyme was shown to incise damaged DNA at pyrimidine sites. The enzyme incised DNA damaged with UV light, ionizing radiation, osmium tetroxide, potassium permanganate, and hydrogen peroxide at cytosine and thymine sites. The substrate specificity of the calf thymus endonuclease was compared to that of Escherichia coli endonuclease III. Similar pyrimidine base damage specificities were found for both enzymes. These results define a highly conserved class of enzymes present in both procaryotes and eucaryotes that may mediate an important role in the repair of oxidative DNA damage.

Animals↗
PubMed2002

Characterization of substrate specificity of plant FatA and FatB acyl-ACP thioesterases.

The specificity of plant acyl-acyl carrier protein (ACP) thioesterases is the major determinant of the chain length and level of saturated fatty acids found in most plant tissues. Although these enzymes have been previously characterized from a number of sources, information on kinetic parameters for a wide range of substrates with cloned enzymes is lacking. In the present study the substrate specificity of recombinant FatA thioesterase isoforms from Arabidopsis (AtFatA) and coriander (CsFatA) and FatB from Arabidopsis (AtFatB) have been re-examined with a comprehensive range of substrates including 14:1-ACP and 16:1-ACP. AtFatA displayed the highest catalytic efficiencies (kcat/Km) towards oleoyl-ACP with activities at least 20-fold lower for all other tested substrates and 75-fold lower with palmitoyl-ACP. Both chain length and double bond presence strongly influenced kcat of FatA with minor influence on Km. Arabidopsis FatB substrate specificity was found to differ from previous reports and this difference could be attributed to the influence of ACP structure. FatB activity with palmitoyl-ACP was 2.5-fold higher and the ratio of 16:0-ACP/14:0-ACP hydrolysis was 6.4-fold higher with spinach ACP compared to E. coli ACP. Additionally, the influence of amino acid domains from both AtFatA and AtFatB on their substrate specificity was studied by utilizing a domain-swapping approach. The characterization of the resulting chimeric enzymes pointed to the N-terminus as a determinant of the substrate specificity for both FatA and FatB acyl-ACP thioesterases.

Arabidopsis↗
PubMed1994

Construction of aminotransferase chimeras and analysis of their substrate specificity.

Escherichia coli aspartate aminotransferase (AspAT) and E. coli aromatic amino acid aminotransferase (AroAT) have almost identical and high activities toward acidic amino acid substrates. AroAT also has high activity toward aromatic amino acid substrates. The two proteins have 44% amino acid sequence homology. In order to study the mechanism responsible for the different substrate specificities of these aminotransferases, chimeric enzymes of AspAT and AroAT were constructed using homologous recombination in E. coli cells. Five chimeric enzymes were obtained, even though the nucleotide sequence homology between the two parent enzymes was as low as about 50%. The yields of the legitimate chimeric genes were related to the lengths of the homologous region between the two parent genes. Homologous recombination occurred in the region where more than eight nucleotides out of ten were identical. The substrate specificity of the chimeric enzymes suggest that not only the amino acid residues in the active site but also those distant from the active site contribute to the substrate specificity of the parental aminotransferases.

Amino Acid Sequence↗
PubMed1996

Substrate specificity of natural variants and genetically engineered intermediates of Bacillus lentus alkaline proteases.

Three natural variants of subtilisin lentus could be differentiated by their amino acid sequence and their specific activity with low molecular weight peptide substrates of the type sAAPFpNA. The variants had amino acid exchanges in five, respective six positions of their amino acid sequence, four of which are located in the substrate loop of the enzyme (positions 92 - 102). Variants of one type of highly alkaline subtilisin (subtilisin 309) were made by site directed mutagenesis, each containing one of the corresponding amino acid exchanges. These intermediate forms were tested for activity, pH-dependence and substrate specificity. The changes in substrate affinity were relatively small for substrates with different amino acids as P1 residue. The differences in activity on peptide-substrates could be related primarily to a single amino acid substitution in the S4 substrate binding pocket in position 102. With substrate variations in the P3 amino acid residue, changes in k(cat) and K(m) revealed the importance of the charged amino acid exchanged between subtilisin 309 and BLAP. By these experiments an interaction of amino acid position 101 and the P3 residue of the substrate could be demonstrated. The substitution of two differently charged amino acids in the substrate binding region resulted in an unchanged pH-profile of the natural enzyme. With the single exchange intermediates differences in the pH-profile could be found, depending on the substrate tested: a characteristic change was observed with casein as substrate, no such change occurred with hemoglobin.

Alkalies↗