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PubMed1997

Factors controlling the substrate specificity of peroxidases: kinetics and thermodynamics of the reaction of horseradish peroxidase compound I with phenols and indole-3-acetic acids.

The rates of oxidation of reducing substrates by heme peroxidases have previously been thought to be controlled only by their ease of oxidation. In the present study, we have compared the kinetics and thermodynamics of the oxidation of indole-3-acetic acid and derivatives and of phenols by horseradish peroxidase. Different dependencies of the reaction rates on the thermodynamic driving force reveal substrate specificity controlled by the enzyme-substrate complexes dissociation constants (Michaelis-Menten constants) and by the reorganization energies of electron-transfer within those complexes.

Catalysis↗
PubMed1976

Substrate specificity of the L-serine O-sulphate degrading activities of Pseudomonas FR.

1. Two enzyme systems obtained from Pseudomonas FR capable of catalysing the alphabeta-elimination of L-serine O-sulphate exhibit a wide range of substrate specificity. Greatest activity was exhibited towards beta-substituted serine and cysteine derivatives. Enzyme A shows a marked preference for the L-isomeric form and enzyme B shows a preference for D-isomers. 2. The alternative activities were shown to be properties of the same enzyme by inhibition properties and heat denaturation experiments. 3. The assay of enzyme A by a number of alternative substrates at various stages during its purification confirmed the multi-substrate specificity of the system. 4. Growth of Pseudomonas FR on S-methyl-L-cysteine as the sole carbon source also resulted in the induction of enzyme B. Growth patterns and levels of induced enzyme were similar to those obtained when L-serine O-sulphate was employed in comparable circumstances.

Isoenzymes↗
PubMed1987

Distinguishing among protein kinases by substrate specificities.

In the previous paper, N-methylated peptides were shown to be sensitive probes of substrate conformation within the adenosine cyclic 3',5'-phosphate dependent protein kinase (A-kinase) active site. While it has been shown that other protein kinases will catalyze the phosphorylation of the same peptide sequences as A-kinase, there is as yet little information as to whether the protein kinases differentiate between substrates on the basis of conformation. For this reason, the conformationally restricted N-methylated peptides were used to probe the active site of guanosine cyclic 3',5'-phosphate dependent protein kinase (G-kinase), which is homologous in sequence to [Takio, K., Wade, R. D., Smith, S. B., Krebs, E. G., Walsh, K. A., & Titani, K. (1984) Biochemistry 23, 4207-4218] and which has substrate specificities similar to [Lincoln, T. M., & Corbin, J. D. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3239-3243] those of A-kinase. Although this enzyme appears to bind the peptides in a conformation resembling that of conformation A, it is more able to accommodate backbone methylation than is A-kinase. A peptide substrate at least 700-fold selective for G-kinase over A-kinase was found. Backbone methylation may, therefore, represent a way of making peptide substrates and inhibitors selective for a particular kinase.

Animals↗
PubMed1978

Substrate specificity and mode of action of a cellulase from Aspergillus niger.

The mode of action and substrate specificity of a cellulase purified from Aspergillus niger were examined. The enzyme showed little capacity to hydrolyse highly ordered cellulose, but readily attacked soluble cellulose derivatives and amorphous alkali-swollen cellulose. Activity towards barley glucan and lichenin was greater than with CM-cellulose. Low activity was detected with CM-pachyman (a substituted beta-1,3-glucose polymer) and xylan. Activity towards yeast glucan, mannan, ethlene glycol chitin, glycol chitosan, laminarin, polygalacturonic acid and pectin could not be demonstrated. Cellobiose and p-nitrophenyl beta-D-glucoside were not hydrolysed, whereas the rate of hydrolysis of the higher members of the reduced cellulodextrins increased with chain length. The central bonds of cellotetraosylsorbitol and cellopentaosylsorbitol were the preferred points of clevage. Kinetic data indicated that the specificity region of the cellulase is five glucose units in length. The evidence indicates that the cellulase is an endoglucanase.

Aspergillus niger↗
PubMed1975

Cholesterol oxidase. Further studies of substrate specificity in relation to the analytical characterisation of steroids.

The substrate specificity of cholesterol oxidase has been further examined with respect to the size and shape of the steroid 17beta-side-chain and oxygenation of the nucleus. The relatively unreactive 17-keto-delta5-3beta-hydroxysteroids were converted to satisfactory substrates by formation of the isopentyl- or benzyloximes. The results obtained have been used to illustrate further uses of the enzyme in the gas-phase analytical characterisation and selective oxidation steroids. Oxidations were studied by kinetic experiments, gas-liquid chromatography-mass spectrometry.

17-Ketosteroids↗
PubMed1995

On the levels of enzymatic substrate specificity: implications for the early evolution of metabolic pathways.

The most frequently invoked explanation for the origin of metabolic pathways is the retrograde evolution hypothesis. In contrast, according to the so-called "patchwork" theory, metabolism evolved by the recruitment of relatively inefficient small enzymes of broad specificity that could react with a wide range of chemically related substrates. In this paper it is argued that both sequence comparisons and experimental results on enzyme substrate specificity support the patchwork assembly theory. The available evidence supports previous suggestions that gene duplication events followed by a gradual neoDarwinian accumulation of mutations and other minute genetic changes lead to the narrowing and modification of enzyme function in at least some primordial metabolic pathways.

Bacteria↗
PubMed1986

Substrate specificity and characterization of rat liver p-nitrophenol, 3 alpha-hydroxysteroid and 17 beta-hydroxysteroid UDP-glucuronosyltransferases.

Purified preparations of rat liver 17-hydroxysteroid, 3-hydroxyandrogen and p-nitrophenol (3-methylcholanthrene-inducible) UDP-glucuronosyltransferases were further characterized as to their substrate specificities, phospholipid-dependency and physical properties. The two steroid UDP-glucuronosyltransferases were shown to exhibit strict stereospecificity with respect to the conjugation of steroids and bile acids. These enzymes have been renamed 17 beta-hydroxysteroid and 3 alpha-hydroxysteroid UDP-glucuronosyltransferase to reflect this specificity for important endogenous substrates. An endogenous substrate has not yet been identified for the p-nitrophenol (3-methylcholanthrene-inducible) UDP-glucuronosyltransferase. The steroid UDP-glucuronosyltransferase activities were dependent on phospholipid for maximal catalytic activity. Complete delipidation rendered the UDP-glucuronosyltransferases inactive, and enzymic activity was not restored when phospholipid was added to the reaction mixture. After partial delipidation, phosphatidylcholine was the most efficient phospholipid for restoration of enzymic activity. Partial delipidation also altered the kinetic parameters of the 3 alpha-hydroxysteroid UDP-glucuronosyltransferase. The three purified UDP-glucuronosyltransferases are separate and distinct proteins, with different amino acid compositions and peptide maps generated by limited proteolysis with Staphylococcus aureus V8 proteinase. Some similarity was observed between the amino acid composition and limited proteolytic maps of the steroid UDP-glucuronosyltransferases, suggesting they are more closely related to each other than to the p-nitrophenol UDP-glucuronosyltransferase.

Amino Acids↗
PubMed1977

New formamidase having substrate specificity for o-formylaminoacetophenone in pig liver.

The supernatant (13 000 x g, 20 min) of pig liver homogenate was filtered with Sephadex G-200 and formamidase (aryl-formylamine amidohydrolase, EC 3.5.1.9)activity in each fraction was measured. When formylkynurenine was used as substrate, two peaks of formamidase activity were observed but, with formylaminoacetophenone as substrate, only one peak was observed. Formamidase in the lower molecular weight fraction is known as kynurenine formamidase (FA I), formamidase found here in the higher molecular weight fraction has not been previously reported. This form, designated FA II has been purified about 160-fold from pig liver. The formamidase obtained has substrate specificity for o-formylaminoacetophenone only and could not hydrolyze formylkynurenine. The optimal pH was 8.5 and the Km for o-formylaminoacetophenone was 1.66-10(-3) M. This formamidase was considered to be a new enzyme and was different from FA I in molecular weight and substrate specificity. This new formamidase was present in pig, rabbit and guinea pig liver and not present in rat or mouse liver.

Acetophenones↗
PubMed1999

Substrate specificity studies of Flavobacterium chondroitinase C and heparitinases towards the glycosaminoglycan--protein linkage region. Use of a sensitive analytical method developed by chromophore-labeling of linkage glycoserines using dimethylaminoazobenzenesulfonyl chloride.

Bacterial chondroitinases and heparitinases are potentially useful tools for structural studies of chondroitin sulfate and heparin/heparan sulfate. Substrate specificities of Flavobacterium chondroitinase C, as well as heparitinases I and II, towards the glycosaminoglycan-protein linkage region -HexA-HexNAc-GlcA-Gal-Gal-Xyl-Ser (where HexA represents glucuronic acid or iduronic acid and HexNAc represents N-acetylgalactosamine or N-acetylglucosamine) were investigated using various structurally defined oligosaccharides or oligosaccharide-serines derived from the linkage region. In the case of oligosaccharide-serines, they were labeled with a chromophore dimethylaminoazobenzenesulfonyl chloride (DABS-Cl), which stably reacted with the amino group of the serine residue and rendered high absorbance for microanalysis. Chondroitinase C cleaved the GalNAc bond of the pentasaccharides or hexasaccharides derived from the linkage region of chondroitin sulfate chains and tolerated sulfation of the C-4 or C-6 of the GalNAc residue and C-6 of the Gal residues, as well as 2-O-phosphorylation of the Xyl residue. In contrast, it did not act on the GalNAc-GlcA linkage when attached to a 4-O-sulfated Gal residue. Heparitinase I cleaved the innermost glucosaminidic bond of the linkage region oligosaccharide-serines of heparin/heparan sulfate irrespective of substitution by uronic acid, whereas heparitinase II acted only on the glucosaminidic linkages of the repeating disaccharide region, but not on the innermost glucosaminidic linkage. These defined specificities of chondroitinase C, as well as heparitinases I and II, will be useful for preparation and structural analysis of the linkage oligosaccharides.

Bacterial Proteins↗
PubMed2002

Structure determinants of substrate specificity of hydroxynitrile lyase from Manihot esculenta.

Tryptophan 128 of hydroxynitrile lyase of Manihot esculenta (MeHNL) covers a significant part of a hydrophobic channel that gives access to the active site of the enzyme. This residue was therefore substituted in the mutant MeHNL-W128A by alanine to study its importance for the substrate specificity of the enzyme. Wild-type MeHNL and MeHNL-W128A showed comparable activity on the natural substrate acetone cyanohydrin (53 and 40 U/mg, respectively). However, the specific activities of MeHNL-W128A for the unnatural substrates mandelonitrile and 4-hydroxymandelonitrile are increased 9-fold and approximately 450-fold, respectively, compared with the wild-type MeHNL. The crystal structure of the MeHNL-W128A substrate-free form at 2.1 A resolution indicates that the W128A substitution has significantly enlarged the active-site channel entrance, and thereby explains the observed changes in substrate specificity for bulky substrates. Surprisingly, the MeHNL-W128A--4-hydroxybenzaldehyde complex structure at 2.1 A resolution shows the presence of two hydroxybenzaldehyde molecules in a sandwich type arrangement in the active site with an additional hydrogen bridge to the reacting center.

Aldehyde-Lyases↗
PubMed1981

On the substrate specificity of cathepsin L.

A view is given on the maximal hydrolysis of proteins by cathepsin L (EC 3.4.22.15) in dependence on the pH. The overall degradation of several proteins at pH values lower than pH 6.0 implies a very broad specificity, whereas at pH 7.0 and 7.5 cathepsin L seems to act on proteins cleaving only restricted specific peptide bonds. Some kinetic constants are given for the three synthetic substrates of cathepsin L which are known so far: Bz-Arg-NH2, Z-Lys-OPhNO2 and Z-Phe-Arg-NMec. They cannot be used as completely specific substrates of cathepsin L, because all of them are hydrolysed by cathepsin B and also other proteinases.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1990

Further characterization of the substrate specificity of a TRH hydrolysing pyroglutamate aminopeptidase from guinea-pig brain.

In this study the substrate specificity of a pyroglutamate aminopeptidase from synaptosomal membranes of guinea-pig brain was investigated. The enzyme was found to be specific for tripeptides, tripeptide-amides and tetrapeptides which possess the N-terminal sequence Glp-His and as such is specific for Thyrotropin Releasing Hormone or only very closely related peptides. The enzyme was found not to hydrolyse a number of analogues of Thyrotropin Releasing Hormone which have been shown to have therapeutical value in certain neuronal disorders.

Aminopeptidases↗
PubMed1988

On the substrate specificity of cytochrome P450IIIA1.

The instability of the solubilized/purified form, the lack of catalytic activity of the stabilized, macrolide-complexed form, and the compromised catalytic activity of the decomplexed form of steroid-inducible cytochrome P450IIIA1 motivated further investigations of the substrate specificity of this isozyme. A major complementary goal was to identify reactions utilizable as sensitive, specific diagnostic probes for the detection and partial characterization of this isozyme in tissues for which isolation and purification are not practical (e.g., extrahepatic, embryonic tissues, etc.). The approach utilized a combination of a specific, purified inducer, specific inhibitors including triacetyloleandomycin and inhibitory antibodies, and diagnostic probe substrates including the phenoxazone ethers, testosterone, warfarin, 2-acetylaminofluorene, estradiol-17 beta and benzo[a]pyrene. The results obtained indicated that steroid-inducible, rat hepatic P450IIIA1 would catalyze minimal or no O-dealkylation of methoxy-, ethoxy- or pentoxyphenoxazone but catalyzed rapid O-debenzylation of benzyloxyphenoxazone. Hydroxylation of testosterone was specific for the beta face of the molecule at the 2-, 6-, 15- and 16-positions with no detectable conversion to androstenedione and minimal hydroxylation on the alpha face. Both the R- and S-enantiomers of warfarin were attacked at positions 9 and 10, and these reactions appeared to be specific to isozymes of the IIIA family. Aromatic hydroxylation of estradiol-17 beta was efficiently catalyzed, particularly at the 2-position. Hydroxylations of 2-acetylaminofluorene at positions 5 and 7 were catalyzed at relatively rapid rates, but N-hydroxylation of the same substrate was not catalyzed effectively. Hydroxylation of benzo[a]pyrene occurred preferentially at carbon 3 with much lesser activity at carbon 9 and little or no detectable attack at positions 7 or 1. The results indicated that the 2 beta- and 15 beta-hydroxylation of testosterone and the 10-hydroxylation of warfarin would serve as the most useful probes thus far available for detection of the presence of functional P450IIIA1 isozymes in tissues for which isolation and purification are impractical. The results also indicated a very broad, yet selective substrate specificity for the steroid-inducible P450IIIA1.

2-Acetylaminofluorene↗
PubMed2003

Substrate specificity of the violaxanthin de-epoxidase of the primitive green alga Mantoniella squamata (Prasinophyceae).

The substrate specificity of the enzyme violaxanthin de-epoxidase (VDE) of the primitive green alga Mantoniella squamata (Prasinophyceae) was tested in in vitro enzyme assays employing the following xanthophyll mono-epoxides: antheraxanthin (Ax), diadinoxanthin (Ddx), lutein-epoxide (LE), cryptoxanthin-epoxide (CxE), 9- cis neoxanthin (cNx), all- trans neoxanthin (Nx), and xanthophyll di-epoxides: 9- cis violaxanthin (cVx), all- trans violaxanthin (Vx), cryptoxanthin-di-epoxide (CxDE). The data presented in this study show that the VDE of M. squamata not only exhibits a low affinity for the mono-epoxide Ax, as has been reported by R. Frommolt et al. (2001, Planta 213:446-456), but has a reduced substrate affinity for the mono-epoxides Ddx, LE, CxE, and Nx as well. On the other hand, xanthophylls with a second epoxy-group (Vx, CxDE) can be de-epoxidized with a higher efficiency. Such a preference for xanthophyll di-epoxides cannot be observed for the higher-plant VDE, where, in general, no marked differences in the pigment de-epoxidation rates between xanthophyll mono- and di-epoxides are visible. Despite this substantial difference between the VDEs of M. squamata and S. oleracea there are also features common to both enzymes. Neither VDE is able to convert xanthophylls with a 9- cis configuration in the acyclic polyene chain and both rely on substrates in the all- trans configuration. Both enzymes furthermore exhibit a dependence of enzyme activity on the polarity of the substrate. Highly polar (Nx) or non-polar (CxE) xanthophylls are de-epoxidized with greatly reduced rates in comparison to substrates with an intermediate polarity (Vx, Ax, LE, Ddx). This dependence on substrate polarity becomes more obvious when the higher-plant VDE is examined, as the substrate affinity of the VDE of M. squamata is more strongly influenced by the existence or absence of a second epoxy-group. In summary, the data presented in this study underline the fact that different VDEs, although in general catalyzing the same reaction sequence, are functionally diverse.

Chlorophyta↗
PubMed2000

Genetic and biochemical characterization of a protein phosphatase with dual substrate specificity in Streptomyces coelicolor A3(2).

A gene encoding a protein phosphatase (SppA) with a phosphoesterase motif, which was predicted by the genome project of the Gram-positive bacterium Streptomyces coelicolor A3(2), was cloned by PCR in pET32a(+) and expressed in Escherichia coli. SppA fused to thioredoxin (TRX-SppA) showed distinct heat-stable phosphatase activity toward p-nitrophenyl phosphate with optimal pH 8.0 and optimal temperature 55 degrees C. Mn2+ greatly enhanced enzyme activity, as is found with other protein Ser/Thr phosphatases. TRX-SppA was not inhibited by sodium orthovanadate or okadaic acid, both of which are known to be specific inhibitors of protein phosphatases. TRX-SppA showed phosphatase activity toward not only phosphoThr (pThr) and pTyr but also oligopeptides containing pSer, pThr, and pTyr, indicating that SppA is a protein phosphatase with dual substrate specificity. Disruption of the chromosomal sppA gene resulted in severe impairment of vegetative growth. All of these observations show that SppA, a protein phosphatase with dual specificity, plays an important, but not essential, role in vegetative growth of S. coelicolor A3(2). The presence of a single copy of sppA in all the 13 Streptomyces species examined, as determined by Southern hybridization, suggests a common role of SppA in general in Streptomyces species.

Amino Acid Sequence↗
PubMed1995

Substrate specificity of alpha-L-arabinofuranosidase from Bacillus subtilis 3-6 toward arabinofurano-oligosaccharides.

The substrate specificity of alpha-L-arabinofuranosidase (EC 3.2.1.55) from Bacillus subtilis 3-6 was explored using methyl 2-O-, methyl 3-O-, and methyl 5-O-alpha-L-arabinofuranosyl-alpha-L-arabinofuranosides, and methyl 3,5-di-O-alpha-L-arabinofuranosyl-alpha-L-arabinofuranoside. The enzyme hydrolyzed the methyl alpha-L-arabinofuranobiosides to arabinose and methyl alpha-L-arabinofuranoside in the order of (1-->2)- > (1-->3)- > (1-->5)-linkages. The enzyme hydrolyzed the (1-->3)-linkage more than the (1-->5)-linkage of the methyl alpha-L-arabinofuranotrioside.

Bacillus subtilis↗
PubMed1990

[Substrate specificity of glycogen synthase from rabbit skeletal muscles].

Nitrous bases were shown to play an essential role in the specificity of active and adenyl nucleotide binding sites. Pyrimidine base determines the substrate specificity of rabbit skeletal muscle glycogen synthase; a crucial role in this process is ascribed to the lactam fragment of the pyrimidine cycle. The 2-oxo group was also shown to be involved in substrate binding. The adenyl nucleotide binding site interacts only with 6-aminopurine derivatives. A negative interaction was found between the enzyme active center and the adenyl nucleotide binding site.

Adenine Nucleotides↗
PubMed1983

Substrate specificity of cyclic nucleotide phosphodiesterase from beef heart and from Dictyostelium discoideum.

The substrate specificity of beef heart phosphodiesterase activity and of the phosphodiesterase activity at the cell surface of the cellular slime mold Dictyostelium discoideum has been investigated by measuring the apparent Km and maximal velocity (V) of 24 derivatives of adenosine 3',5'-monophosphate (cAMP). Several analogs have increased Km values, but unaltered V values if compared to cAMP; also the contrary (unaltered Km and reduced V) has been observed, indicating that binding of the substrate to the enzyme and ring opening are two separate steps in the hydrolysis of cAMP. cAMP is bound to the beef heart phosphodiesterase by dipole-induced dipole interactions between the adenine moiety and an aromatic amino acid, and possibly by a hydrogen bond between the enzyme and one of the exocyclic oxygen atoms; a cyclic phosphate ring is not required to obtain binding. cAMP is bound to the slime mold enzyme via a hydrogen bond at the 3'-oxygen atom, and probably via a hydrogen bond with one of the exocyclic oxygen atoms. A cyclic phosphate ring is necessary to obtain binding to the enzyme. A specific interaction (polar or hydrophobic) between the base moiety and the enzyme has not been demonstrated. A negative charge on the phosphate moiety is not required for binding of cAMP to either enzyme. The catalytic reaction in both enzymes is restricted to the phosphorus atom and to the exocyclic oxygen atoms. Substitution of the negatively charged oxygen atom by an uncharged dimethylamino group in axial or equatorial position renders the compound non-hydrolyzable. Substitution of an exocyclic oxygen by a sulphur atom reduces the rate of the catalytic reaction about 100-fold if sulphur is placed in axial position and more than 10000-fold if sulphur is placed in equatorial position. A reaction mechanism for the enzymatic hydrolysis of cAMP is proposed.

3',5'-Cyclic-AMP Phosphodiesterases↗