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PubMed1999

Comparison of the stability and substrate specificity of purified peroxisomal 3-oxoacyl-CoA thiolases A and B from rat liver.

The specific activities and substrate specificities of 3-oxoacyl-CoA thiolase A (thiolase A) purified from normal rat liver peroxisomes and 3-oxoacyl-CoA thiolase B (thiolase B) isolated from livers of rats treated with the peroxisome proliferator clofibrate were virtually identical. The enzymes could be distinguished by their N-terminal amino acid sequences, their isoelectric points and their stability, the latter being higher for thiolase A. Contrary to thiolase B, which showed a marked cold lability in the presence of KCl by dissociating into monomers with poor activity, thiolase A retained its full activity and its homodimeric structure under these conditions.

Acetyl-CoA C-Acyltransferase↗
PubMed1981

Cardiac sarcolemmal and sarcoplasmic reticulum membrane vesicles exhibit distinctive (Ca-Mg)-ATPase substrate specificities.

The nucleoside 5'-triphosphate (NTP) substrate specificities for Ca-stimulated ATPase and ATP-dependent Ca2+ uptake activities have been examined in cardiac sarcolemma (SL) and sarcoplasmic (SR) membrane vesicles. The results indicate that SL membrane vesicles exhibit a much narrower range of NTP substrate specificities than SR membranes. In SR membrane vesicles, the Ca-stimulated Mg-dependent hydrolysis of ATP and dATP occurred at nearly equivalent rates, whereas the rates of hydrolysis of GTP, ITP, CTP, and UTP ranged from 16-33% of that for ATP. All of the above nucleotides also supported Ca2+ transport into SR vesicles; dATP was somewhat more effective than ATP while GTP, ITP, CTP, and UTP ranged from 28-30% of the activity for ATP. In the presence of oxalate, the initial rate of Ca accumulation with dATP was 4-fold higher than for ATP, whereas the activity for GTP, ITP, CTP, and UTP ranged from 35-45% of that for ATP. For the SL membranes, Ca-activated dATP hydrolysis occurred at 60% of the rate for ATP; GTP, ITP, CTP, and UTP were hydrolyzed by the SL preparations at only 7-9% of the rate for ATP. NTP-dependent Ca2+ uptake in SL membranes was supported only by ATP and dATP, with dATP 60% as effective as ATP. GTP, ITP, CTP, and UTP did not support the transport of Ca2+ by SL vesicles. The results indicate that the SL and SR membranes contain distinctly different ATP-dependent Ca2+ transport systems.

Animals↗
PubMed2002

Involvement of a nine-residue loop of streptokinase in the generation of macromolecular substrate specificity by the activator complex through interaction with substrate kringle domains.

The selective deletion of a discrete surface-exposed epitope (residues 254-262; 250-loop) in the beta domain of streptokinase (SK) significantly decreased the rates of substrate human plasminogen (HPG) activation by the mutant (SK(del254-262)). A kinetic analysis of SK(del254-262) revealed that its low HPG activator activity arose from a 5-6-fold increase in K(m) for HPG as substrate, with little alteration in k(cat) rates. This increase in the K(m) for the macromolecular substrate was proportional to a similar decrease in the binding affinity for substrate HPG as observed in a new resonant mirror-based assay for the real-time kinetic analysis of the docking of substrate HPG onto preformed binary complex. In contrast, studies on the interaction of the two proteins with microplasminogen showed no difference between the rates of activation of microplasminogen under conditions where HPG was activated differentially by nSK and SK(del254-262). The involvement of kringles was further indicated by a hypersusceptibility of the SK(del254-262).plasmin activator complex to epsilon-aminocaproic acid-mediated inhibition of substrate HPG activation in comparison with that of the nSK.plasmin activator complex. Further, ternary binding experiments on the resonant mirror showed that the binding affinity of kringles 1-5 of HPG to SK(del254-262).HPG was reduced by about 3-fold in comparison with that of nSK.HPG . Overall, these observations identify the 250 loop in the beta domain of SK as an important structural determinant of the inordinately stringent substrate specificity of the SK.HPG activator complex and demonstrate that it promotes the binding of substrate HPG to the activator via the kringle(s) during the HPG activation process.

Base Sequence↗
PubMed1978

Metabolism of trichothecene mycotoxins. II. Substrate specificity of microsomal deacetylation of trichothecenes.

The substrate specificity of microsomal nonspecific carboxyesterase [EC 3.1.1.1] from rabbit and rat livers was studied in vitro by using seven (A)-type and six (B)-type 12,13-epoxytrichothecene mycotoxins. The C-4 acetyl residues of diacetoxyscirpenol, T-2 toxin, monoacetylnivalenol (fusarenon-X), and diacetylnivalenol were selectively hydrolyzed by the microsomal esterase to yield the corresponding C-4-deacetylated metabolites: monoacetoxyscirpenol, HT-2 toxin, nivalenol, and 15-acetylnivalenol, respectively. The C-3 acetyl group of monoacetyldeoxynivalenol and the C-8 acetyl group of tetraacetoxyscirpen were also deacetylated. Triacetoxyscirpen gave rise to two unidentified metabolites, which may include a C-4-deacetylated product. 8-Hydroxydiacetoxyscirpenol (neosolaniol), HT-2 toxin, acetyl-T-2 toxin and tetraacetylnivalenol were unaffected by this type of hydrolysis. It follow from these results that the C-4 acetyl residue is hydrolyzed by the microsomal carboxyesterase and substituents at C-3 and C-8 contribute to the selective enzymatic hydrolysis of the C-4 acetyl residue of trichothecenes. Kinetic analysis showed that rabbit microsomal esterase possessed a high affinity for (A)-type trichothecenes such as T-2 toxin and diacetoxyscirpenol, and that of rat microsomes possessed a high affinity for (B)-type trichothecenes such as monoacetylnivalenol (fusarenon-X). The significance of this specific deacetylation reaction is discussed in relation to the biological activity of the trichothecene derivatives as revealed by their inhibitory effect on protein synthesis in rabbit reticulocytes.

Animals↗
PubMed2004

Val216 decides the substrate specificity of alpha-glucosidase in Saccharomyces cerevisiae.

Differences in the substrate specificity of alpha-glucosidases should be due to the differences in the substrate binding and the catalytic domains of the enzymes. To elucidate such differences of enzymes hydrolyzing alpha-1,4- and alpha-1,6-glucosidic linkages, two alpha-glucosidases, maltase and isomaltase, from Saccharomyces cerevisiae were cloned and analyzed. The cloned yeast isomaltase and maltase consisted of 589 and 584 amino acid residues, respectively. There was 72.1% sequence identity with 165 amino acid alterations between the two alpha-glucosidases. These two alpha-glucosidase genes were subcloned into the pKP1500 expression vector and expressed in Escherichia coli. The purified alpha-glucosidases showed the same substrate specificities as those of their parent native glucosidases. Chimeric enzymes constructed from isomaltase by exchanging with maltase fragments were characterized by their substrate specificities. When the consensus region II, which is one of the four regions conserved in family 13 (alpha-amylase family), is replaced with the maltase type, the chimeric enzymes alter to hydrolyze maltose. Three amino acid residues in consensus region II were different in the two alpha-glucosidases. Thus, we modified Val216, Gly217, and Ser218 of isomaltase to the maltase-type amino acids by site-directed mutagenesis. The Val216 mutant was altered to hydrolyze both maltose and isomaltose but neither the Gly217 nor the Ser218 mutant changed their substrate specificity, indicating that Val216 is an important residue discriminating the alpha-1,4- and 1,6-glucosidic linkages of substrates.

Amino Acid Sequence↗
PubMedDate not supplied

[Detection, in synaptic membranes of proteins, specific substrates for endogenous phosphorylation in the presence of GTP].

Using the polyacrylamide gel electrophoresis (PAGE) of the synaptic membranes of proteins isolated from the rat brain cortex it is shown that on phosphorylation in the presence of [gamma-33P] GTP (5 and 10 microM) and 10-20-fold excess of unlabelled ATP the phosphorylation of protein with molecular weights of 41,000 and 49,000 Dalton greatly increased but the labelling of proteins with molecular weights of 54000 and 30,000 Dalton strongly decreased or was completely abolished. The addition of unlabelled ATP practically does not change the phosphorylation of the bands (proteins) with molecular weights of 86,000, 82,000, 46,000 Dalton and weakly decreased when labelling proteins with molecular weight of 59,000. The results obtained permit suggesting the existence of proteins--substrates of specific phosphorylation with GTP in synaptic membranes.

Adenosine Triphosphate↗
PubMed2002

Studies into factors contributing to substrate specificity of membrane-bound 3-ketoacyl-CoA synthases.

We are interested in constructing a model for the substrate-binding site of fatty acid elongase-1 3-ketoacyl CoA synthase (FAE1 KCS), the enzyme responsible for production of very long chain fatty acids of plant seed oils. Arabidopsis thaliana and Brassica napus FAE1 KCS enzymes are highly homologous but the seed oil content of these plants suggests that their substrate specificities differ with respect to acyl chain length. We used in vivo and in vitro assays of Saccharomyces cerevisiae-expressed FAE1 KCSs to demonstrate that the B. napus FAE1 KCS enzyme favors longer chain acyl substrates than the A. thaliana enzyme. Domains/residues responsible for substrate specificity were investigated by determining catalytic activity and substrate specificity of chimeric enzymes of A. thaliana and B. napus FAE1 KCS. The N-terminal region, excluding the transmembrane domain, was shown to be involved in substrate specificity. One chimeric enzyme that included A. thaliana sequence from the N terminus to residue 114 and B. napus sequence from residue 115 to the C terminus had substrate specificity similar to that of A. thaliana FAE1 KCS. However, a K92R substitution in this chimeric enzyme changed the specificity to that of the B. napus enzyme without loss of catalytic activity. Thus, this study was successful in identifying a domain involved in determining substrate specificity in FAE1 KCS and in engineering an enzyme with novel activity.

Acetyltransferases↗
PubMed1986

Genetic analysis of a relaxed substrate specificity aromatic ring dioxygenase, toluate 1,2-dioxygenase, encoded by TOL plasmid pWW0 of Pseudomonas putida.

Toluate 1,2-dioxygenase is the first enzyme of a meta-cleavage pathway for the oxidative catabolism of benzoate and substituted benzoates to Krebs cycle intermediates that is specified by TOL plasmid pWW0 of Pseudomonas putida. A collection of derivatives harbouring Tn1000 insertions and defective in toluate dioxygenase have been isolated from pPL392, a pBR322-based hybrid plasmid carrying the TOL plasmid meta-cleavage pathway operon. In parallel, a series of N-methyl-N'-nitro-N-nitro-soguanidine-induced mutant plasmids defective in this enzyme activity were isolated from pNM72, a pKT231-based hybrid plasmid carrying the same operon. Pairs of mutant plasmids, consisting of one Tn1000 derivative and one nitrosoguanidine-induced derivative, were used for complementation analysis of toluate dioxygenase in Escherichia coli recA bacteria, in which the formation of 2-hydroxymuconic semialdehyde from benzoate was examined. Four cistrons for toluate 1,2-dioxygenase were thus identified. DNA fragments containing nitrosoguanidine-induced mutant cistrons plus the other meta-cleavage operon genes were cloned into pOT5, an R388-based vector, and complementation tests between different nitrosoguanidine-induced mutant cistrons were carried out in Pseudomonas putida cells, this time scoring for growth on p-toluate. This analysis also identified four cistrons. Examination of the products of these cistrons, by means of E. coli minicells containing pPL392 or its Tn1000 insertion derivatives, indicated that the first two cistrons of the operon comprise a single gene, xylX, which encodes a 57 kilodalton protein, and that the third cistron, xylY, encodes a 20 kilodalton protein.

DNA Restriction Enzymes↗
PubMed1985

The aromatization of cyclohexanecarboxylic acid to hippuric acid: substrate specificity and species differences.

The ability to convert cyclohexanecarboxylic acid to hippuric acid has been studied in liver from guinea pigs, rabbits, rats and mice using a gas chromatographic - mass spectrometric method employing selected ion monitoring. Guinea pig liver showed the highest activity, giving values double of those found in rabbit liver and five times those in rat liver. Only very weak activity was found in mouse liver. (Hydroxymethyl)cyclohexane, cyclohexanealdehyde and alpha-hydroxyethylcyclohexane, which are structurally related to cyclohexanecarboxylic acid but lack the carboxyl group, were not aromatized by guinea pig liver mitochondria. This finding indicates that the carboxyl group is essential for aromatization. Absence of aromatization was also found with the homologs cyclohexaneacetic acid and cyclohexanepropionic acid and with the di-acids trans-1,2- and trans-1,4-cyclohexanedicarboxylic acid. The effect of a methyl group in cyclohexanecarboxylic acid depended on its position. 2-Methyl-1-cyclohexanecarboxylic acid was not aromatized, however the 3- and 4-methyl derivatives underwent aromatization and subsequent conjugation with glycine. The rates of formation of m-methyl- and p-methylhippuric acid were 16% and 9%, respectively, of that found for hippuric acid from cyclohexanecarboxylic acid (8.0 nmol/min/mg protein).

Animals↗
PubMed1986

Substrate specificities of lipases from corn and other seeds.

Lipases from several seed species were shown to be relatively specific on triacylglycerols containing the major fatty acid components of the storage triacylglycerols in the same species. In a direct comparison using individual triacylglycerol as well as mixed triacylglycerol preparations, highest activities were observed in corn lipase on trilinolein and triolein, castor bean lipase on triricinolein, rapeseed lipase on trierucin, and elm seed lipase on tricaprin. This pattern of fatty acyl specificity was also observed on diacylglycerols, monoacylglycerols, and fatty acyl 4-methylumbelliferone, although the pattern became less distinct. The seed lipases were inactive on lecithins. Corn lipase was more active on tri- than di- or monolinolein, and released linoleic acids from both primary and secondary positions. As judged from the kinetics of hydrolysis of rac-glyceryl-2,3-stearate-1-oleate and rac-glyceryl-1,3-stearate-2-oleate, and of trilinolein and dilinoleins, corn lipase exerted some degree of preference in releasing fatty acid from the primary than the secondary position of a triacylglycerol. At the primary position, corn lipase was more active on oleyl ester than stearyl ester.

Animals↗
PubMed1990

Mode of action and substrate specificity of a purified exo-1,4-beta-D-glucosidase cloned from the cellulolytic bacterium Ruminococcus albus AR67.

A gene encoding exo-1,4-beta-D-glucosidase, from Ruminococcus albus AR67, was cloned in Escherichia coli, restriction mapped, and shown to be expressed from sequences within the insert that function as a promoter in E. coli. The cloned enzyme was located predominantly in the cytoplasm (40%) and attached to insoluble cell components (48%). After purification to homogeneity, the enzyme (Mr = 64,000, monomeric) was specific for substrates with beta-D-glucopyranosyl configuration and was inactive against alpha-glucosides, lactosides and xylosides. Km values of the enzyme decreased with increasing chain length (G2-G5). Glucose was the major product of hydrolysis from cellodextrins. Preference for longer chain cellodextrins is consistent with exo-1,4-beta-D-glucan glucohydrolase mode of action [E.C. 3.2.1.74].

Chromatography, Gel↗