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At least 883 records · Page 49Linked to original sources
PubMed1992

The study on phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis: synthesis of homogeneous substrates, substrate specificity and other properties.

The properties of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis were studied in detail. The enzyme was extremely thermostable in 0.1% bovine serum albumin and retained 73% of its activity at 100 degrees C for 10 min, while it was labile in the absence of albumin. The enzymatic activity was inhibited by HgCl2 or p-chloromercuriphenylsulfonic acid and restored by dithiothreitol. The kinetic parameters (Km and Vmax) of PI-PLC were determined for the mixed micelle of yeast phosphatidylinositol (PI)/Triton X-100 or sodium deoxycholate. Four PIs having different acyl chains: dilauroylphosphatidylinositol (DLPI), dimyristoylphosphatidylinositol (DMPI), dipalmitoylphosphatidylinositol (DPPI) and dioleoylphosphatidylinositol (DOPI) were synthesized from yeast PI through the processes of deacylation and reacylation, identified by infrared (IR) and Fourier transform nuclear magnetic resonance (FT-NMR) spectra, and subjected to the action of PI-PLC. All the synthetic PIs were hydrolyzed by this enzyme, with DLPI and DMPI being the best substrates. PI-PLC did not catalyze the hydrolysis of the phosphatidylnucleosides 5'-phosphatidylcytidine, 5'-phosphatidyluridine, 5'-phosphatidylthymidine, 5'-phosphatidyladenosine and 5'-phosphatidyl-2'-deoxyadenosine.

Bacillus thuringiensis↗
PubMed1997

Probing the substrate specificity of hepatitis C virus NS3 serine protease by using synthetic peptides.

We probed the substrate specificity of a recombinant noncovalent complex of the full-length hepatitis C virus (HCV) NS3 serine protease and NS4A cofactor, using a series of small synthetic peptides derived from the three trans-cleavage sites of the HCV nonstructural protein sequence. We observed a distinct cleavage site preference exhibited by the enzyme complex. The values of the turnover number (k(cat)) for the most efficient NS4A/4B, 4B/5A, and 5A/5B peptide substrates were 1.6, 11, and 8 min(-1), respectively, and the values for the corresponding Michaelis-Menten constants (Km) were 280, 160, and 16 microM, providing catalytic efficiency values (k(cat)/Km) of 92, 1,130, and 8,300 M(-1) s(-1). An alanine-scanning study for an NS5A/5B substrate (P6P4') revealed that P1 Cys and P3 Val were critical. Finally, substitutions at the scissile P1 Cys residue by homocysteine (Hcy), S-methylcysteine (Mcy), Ala, S-ethylcysteine (Ecy), Thr, Met, D-Cys, Ser, and penicillamine (Pen) produced progressively less efficient substrates, revealing a stringent stereochemical requirement for a Cys residue at this position.

Amino Acid Sequence↗
PubMed2000

Human PC4 is a substrate-specific inhibitor of RNA polymerase II phosphorylation.

The activity of cyclin-dependent protein kinases (cdks) is physiologically regulated by phosphorylation, association with the specific cyclin subunits, and repression by specific cdk inhibitors. All three physiological regulatory mechanisms are specific for one or more cdks, but none is known to be substrate specific. In contrast, synthetic cdk peptide inhibitors that specifically inhibit cdk phosphorylation of only some substrates, "aptamers," have been described. Here, we show that PC4, a naturally occurring transcriptional coactivator, competitively inhibits cdk-1, -2, and -7-mediated phosphorylation of the largest subunit of RNA polymerase II (RNAPII), but it does not inhibit phosphorylation of other substrates of the same kinases. Interestingly, the phosphorylated form of PC4 is devoid of kinase inhibitory activity. We also show that wild-type PC4 but not the kinase inhibitory-deficient mutant of PC4 represses transcription in vivo. Our results point to a novel role for PC4 as a specific inhibitor of RNAPII phosphorylation.

Cell Line↗
PubMed2006

Fold-recognition and comparative modeling of human beta3GalT I, II, IV, V and VI and beta3GalNAcT I: prediction of residues conferring acceptor substrate specificity.

beta3GalTs are type II transmembrane proteins that transfer galactose from UDP-Gal donor substrate to acceptor GlcNAc, GalNAc or Gal in beta1-->3-linkage. beta1-->3-linked galactose have been found to be a part of many glycans like glycosphingolipids, core tetrasaccharide of proteoglycans, type 1 chains. The 3-D structure of none of the beta3GalTs is known to date. In this study, the 3-D structures of human beta3GalT I, II, IV, V, VI and beta3GalNAcT I have been modeled using fold-recognition and comparative modeling methods. Residues that constitute the UDP-Gal binding site have been predicted. The models are able to qualitatively rationalize data from the site-directed mutagenesis experiments reported in the literature. Residues likely to be involved in conferring differential acceptor substrate specificity have been predicted by a combination of specificity determining positions prediction (SDPs) and subsequent mapping on the generated 3-D models.

Amino Acid Sequence↗
PubMed1981

Substrate specificity of the nuclear protein kinase NII from porcine liver. Studies with casein variants.

The substrate specificity of the nuclear protein kinase NII from pig liver was studied using purified casein variants alpha s1B and beta A2 both in the phosphorylated and dephosphorylated form. Chymotryptic peptides of the phosphorylated casein were analyzed by ascending thin-layer chromatography followed by thin-layer electrophoresis. Phosphorylated peptides were detected by autoradiography. The determination of a phosphorylated site in a cluster of three serinees was performed by solid phase Edman degradation of the whole beta A2 casein. Phosphorylated beta A2 casein was by far the best substrate. A threonine at position 41 in the amino acid sequence was found to be predominantly phosphorylated. Dephosphorylated beta A2 casein became rephosphorylated at serine-17 whereas phosphorylated at threonine-41 was greatly reduced. In both sites acidic amino acid side chains are present at the C-terminal side though at different positions suggesting that acidic residues in the vicinity of the serin or threonine residue to be phosphorylated play an important role in the recognition by the nuclear protein kinase NII.

Amino Acid Sequence↗
PubMed2002

Peptide substrate specificities and protein cleavage sites of human endometase/matrilysin-2/matrix metalloproteinase-26.

Human endometase/matrilysin-2/matrix metalloproteinase-26 (MMP-26) is a novel epithelial and cancer-specific metalloproteinase. Peptide libraries were used to profile the substrate specificity of MMP-26 from the P4-P4' sites. The optimal cleavage motifs for MMP-26 were Lys-Pro-Ile/Leu-Ser(P1)-Leu/Met(P1')-Ile/Thr-Ser/Ala-Ser. The strongest preference was observed at the P1' and P2 sites where hydrophobic residues were favored. Proline was preferred at P3, and Serine was preferred at P1. The overall specificity was similar to that of other MMPs with the exception that more flexibility was observed at P1, P2', and P3'. Accordingly, synthetic inhibitors of gelatinases and collagenases inhibited MMP-26 with similar efficacy. A pair of stereoisomers had only a 40-fold difference in K(i)(app) values against MMP-26 compared with a 250-fold difference against neutrophil collagenase, indicating that MMP-26 is less stereoselective for its inhibitors. MMP-26 autodigested itself during the folding process. Two of the major autolytic sites were Leu(49)-Thr(50) and Ala(75)-Leu(76), which still left the cysteine switch sequence (PHC(82)GVPD) intact. This suggests that Cys(82) may not play a role in the latency of the zymogen. Interestingly, inhibitor titration studies revealed that only approximately 5% of the total MMP-26 molecules was catalytically active, indicating that the thiol groups of Cys(82) in the active molecules may be dissociated or removed from the active site zinc ions. MMP-26 cleaved Phe(352)-Leu(353) and Pro(357)-Met(358) in the reactive loop of alpha(1)-proteinase inhibitor and His(140)-Val(141) in insulin-like growth factor-binding protein-1, probably rendering these substrates inactive. Among the fluorescent peptide substrates analyzed, Mca-Pro-Leu-Ala-Nva-Dpa-Ala-Arg-NH(2) displayed the highest specificity constant (30,000/molar second) with MMP-26. This report proposes a working model for the future studies of pro-MMP-26 activation, the design of inhibitors, and the identification of optimal physiological and pathological substrates of MMP-26 in vivo.

Humans↗
PubMed2005

How to broaden enzyme substrate specificity: strategies, implications and applications.

For identification of mutations associated with the broadening of enzyme substrate specificity, three strategies, including directed enzyme evolution, are described for selected examples. Implications concerning enzyme models are highlighted. Applications to the field of biocatalysis are discussed. A bidimensional map for the classification of enzyme activities is suggested so as to improve genome annotations.

Catalysis↗
PubMed1992

[Substrate specificity of phosphoribosyl-aminoimidazole-succinocarboxamide synthetase (SAICAR-synthetase) from Saccharomyces cerevisiae yeast].

The substrate specificity of phosphoribosyl-aminoimidazole-succinocarboxamide-synthetase (SAICAR-synthetase, EC 6.3.2.6) of the yeast Saccharomyces cerevisiae towards a set of carboxyaminoimidazole ribotide (CAIR) analogs with modifications in the imidazole ring, ribose and phosphate moieties, as well as aspartic acid analogs has been studied. It was found, in particular, that: i) the presence of double charged phosphate group, 2'- and 3'-hydroxyl groups in the ribose fragment and of an amino group in the imidazole ring of the CAIR molecule is not the absolute requirement for the enzymatic reaction; ii) 3'-carboxy-1.2.4-triazole analog of CAIR is a competitive inhibitor of the enzyme; iii) 2'-deoxy-CAIR is a substrate for both yeast SAICAR-synthetase and its avian liver and human erythrocyte counterparts. A new method designed to determine the SAICAR-synthetase activity with the help of bifunctional enzymes possessing, in addition to the SAICAR-synthetase activity, also a phosphoribosyl-aminoimidazole-carboxylase activity, is proposed; this method is based on the use of 2'-deoxy-CAIR. Some aspartic acid analogs (L-malic acid, beta-threo-oxy-, and beta-threo-fluoro-aspartic acids and alanosine) are substrates for yeast SAICAR-synthetase. The possible involvement of malate as an alternative substrate for the SAICAR-synthetase reaction in vivo is discussed. The results of a comparative analysis of already established primary structures of yeast, bacterial, human, and chicken SAICAR-synthetases are presented.

Amino Acid Sequence↗
PubMed1985

Investigation on the substrate specificity of human plasmin using tripeptidyl-p-nitroanilide substrates.

The hydrolysis of 35 tripeptidyl-p-nitroanilides was studied with human plasmin and the kinetic parameters were determined. The individual contribution of the various side chains to the kinetic parameters was calculated by regression analysis. Considering Km, substrates having Z-D-Ile-Phe-Lys as well as H-D-Ile-Phe-Lys sequences were found to be the best, while Bz-Ile-Leu-Lys and pGlu-Leu-Lys sequences are the best for kcat. The Km values of substrates protected at N-terminus are lower, their kcat values are higher than those of the unprotected ones with the same sequence.

Amino Acid Sequence↗
PubMed1988

Relaxed thiol substrate specificity of glutathione transferase effected by a non-substrate glutathione derivative.

Rat glutathione transferase 4-4 catalyzed the conjugation of 2-mercaptoethanol with 1-chloro-2,4-dinitrobenzene in the presence of S-methyl-glutathione. The reaction was linearly dependent on enzyme concentration and saturation was seen with respect to both 2-mercaptoethanol and S-methyl-glutathione concentration. High concentrations of S-methyl-glutathione were inhibitory. The results suggest that the natural substrate glutathione has two distinct functions in the normal catalytic reaction, (i) induction of a catalytically competent conformation of the enzyme and (ii) provision of the substrate sulfhydryl group in the reaction catalyzed.

Animals↗
PubMed1980

Gaucher disease. III. Substrate specificity of glucocerebrosidase and the use of nonlabeled natural substrates for the investigation of patients.

A reproducible and convenient method for assaying glucocerebrosidase activity using the natural substrates has been developed. From the insoluble pellet fraction of cultured skin fibroblast homogenates, released glucose was measured enzymically using hexokinase coupled with the glucose-6-phosphate dehydrogenase (G6PD) and nicotinamide adenine dinucleotide phosphate (NADP) system. Optimal enzyme assay conditions required both Triton X-100 and sodium taurocholate, pH 4.8. Glucocerebrosidase activities from three patients with type 1 Gaucher disease were 17.5%, 15.8%, and 11.2% of normal (normal = 198 +/- 14 nmol/hr per mg protein, n = 3). The first patient had normal beta-glucosidase activity with the artificial fluorogenic umbelliferone substrate. Interference with the accuracy of the glucose-dependent assay system by either glycolytic or gluconeogenic enzyme activites was not detected under these experimental conditions, and when substrates with long fatty-acid chain lengths (C = 22) were used, markedly decreased glucocerebrosidase activity occurred in both normal individuals and patients. The apparent Km's for the natural substrates were 0.56 +/- 0.05 mM for controls and 2.2-3.3 mM for Gaucher fibroblasts. These data further support the hypothesis that a structurally altered and catalytically deficient enzyme is synthesized in patients with type 1 Gaucher disease and illustrate the value of the natural substrate in investigating patients.

Cells, Cultured↗
PubMed1988

Substrate specificity for formation of cholesterol ester conjugates from fenvalerate analogues and for granuloma formation.

1. The substrate specificity of microsomal carboxyesterase(s) responsible for the formation of cholesteryl [2R]-2-(4-chlorophenyl) isovalerate from fenvalerate was investigated by incubating mouse kidney microsomes with 14C-cholesterol and the following substrates: fenvalerate isomers, fenvalerate analogues, other pyrethroids, methoprene and cycloprate analogues. Among the four isomers of fenvalerate, only the [2R, alpha S]-isomer yielded a cholesterol ester, being identical with the result obtained in the in vivo study. Some fenvalerate analogues produced cholesterol ester conjugates, but no other pyrethroids nor methoprene produced such conjugates. Some cycloprate analogues gave the corresponding cholesterol ester, the yields of which were dependent on their carbon-chain lengths. 2. Cholesterol ester formation in vitro from these fenvalerate analogues was well correlated with granuloma formation observed when the analogues were given to mice at 3000 ppm for a month. 3. Steroids other than cholesterol were also investigated as acceptors of the acid moiety of the [2R, alpha S]-isomer by incubating solubilized carboxyesterase(s) with the [2R, alpha S]-isomer in the presence of egg lecithin and several steroids. Dehydroisoandrosterone and pregnenolone were found to give the corresponding ester conjugates.

Animals↗
PubMed1985

Mechanism of action of cysteine proteinases: oxyanion binding site is not essential in the hydrolysis of specific substrates.

To study the possible stabilization of the oxyanion of the tetrahedral intermediate formed in the course of the catalyses by cysteine proteinases, papain, chymopapain, papaya peptidase A, and ficin, we synthesized N-(benzyloxycarbonyl)phenylalanylthioglycine O-ethyl ester and compared its hydrolysis with that of the corresponding oxygen ester, a highly specific substrate of the above enzymes. It was found that the substitution of sulfur for the carbonyl oxygen hardly affected the second-order rate constant of acylation and diminished catalytic activity by about 1 order of magnitude in deacylation. These results contrast with those obtained with serine proteinases [Asbóth, B., & Polgár, L. (1983) Biochemistry 22, 117-122], where the hydrolysis of thiono esters could not be detected. From the results the following conclusions can be drawn. Stabilization of the tetrahedral intermediate at an oxyanion binding site is not essential with cysteine proteinases. Therefore, and because of the lack of general base catalysis, cysteine proteinases have a less constrained transition-state structure than serine proteinases.

Anions↗
PubMed1993

Differences in substrate specificities of alpha and beta platelet-derived growth factor (PDGF) receptors. Correlation with their ability to mediate PDGF transforming functions.

Recombinant expression of either the alpha or beta platelet-derived growth factor (PDGF) receptors in 32D hematopoietic cells allows efficient coupling of PDGF with mitogenic and chemotactic signaling pathways inherently expressed by those cells. PDGF-BB stimulation of 32D-alpha R or beta R cells results in anti-P-Tyr recovery of cellular proteins possessing similar as well as distinct phosphotyrosine signals. Comparison of the ability of each receptor to couple with known second messengers revealed that both receptors associated with and/or tyrosine phosphorylated phospholipase C-gamma (PLC gamma) and phosphatidylinositol 3-kinase (p85) with similar stoichiometry. However, the beta platelet-derived growth factor receptor (PDGFR) was significantly more efficient at in vivo tyrosine phosphorylation of GTPase-activating protein (GAP). Similar differences in binding affinity for GAP were observed in NIH/3T3 cells which express both receptors. To quantitate the affinities of each receptor for GAP or PLC gamma, we utilized baculovirus-expressed alpha and beta PDGFRs purified by anti-P-Tyr affinity chromatography. Exposure of immunoblots containing bacterially expressed GAP or PLC gamma to activated alpha or beta PDGF receptors led to a comparable high affinity binding of each receptor to PLC gamma, while the beta PDGFR showed a 5-fold higher binding affinity for GAP. In an effort to correlate differences in their substrate specificities with biological properties of the receptors, we compare their abilities to enhance PDGF-A transforming function in NIH/3T3 cells. Cotransfection of PDGF-A with the alpha PDGFR increased PDGF-A transforming activity by approximately 2-fold. However, cotransfection with a chimeric receptor with the catalytic domain of the beta PDGFR but possessing alpha PDGFR ligand binding properties resulted in 17-fold enhancement of PDGF-A transformation. These findings argue that differences in alpha and beta PDGF receptor substrate specificity in NIH/3T3 fibroblasts correlate with greater transforming activity mediated by the beta PDGFR.

3T3 Cells↗
PubMed2006

Rapid peptide-based screening on the substrate specificity of severe acute respiratory syndrome (SARS) coronavirus 3C-like protease by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Severe acute respiratory syndrome coronavirus (SARS-CoV) 3C-like protease (3CL(pro)) mediates extensive proteolytic processing of replicase polyproteins, and is considered a promising target for anti-SARS drug development. Here we present a rapid and high-throughput screening method to study the substrate specificity of SARS-CoV 3CL(pro). Six target amino acid positions flanking the SARS-CoV 3CL(pro) cleavage site were investigated. Each batch of mixed peptide substrates with defined amino acid substitutions at the target amino acid position was synthesized via the "cartridge replacement" approach and was subjected to enzymatic cleavage by recombinant SARS-CoV 3CL(pro). Susceptibility of each peptide substrate to SARS-CoV 3CL(pro) cleavage was monitored simultaneously by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The hydrophobic pocket in the P2 position at the protease cleavage site is crucial to SARS-CoV 3CL(pro)-specific binding, which is limited to substitution by hydrophobic residue. The binding interface of SARS-CoV 3CL(pro) that is facing the P1' position is suggested to be occupied by acidic amino acids, thus the P1' position is intolerant to acidic residue substitution, owing to electrostatic repulsion. Steric hindrance caused by some bulky or beta-branching amino acids in P3 and P2' positions may also hinder the binding of SARS-CoV 3CL(pro). This study generates a comprehensive overview of SARS-CoV 3CL(pro) substrate specificity, which serves as the design basis of synthetic peptide-based SARS-CoV 3CL(pro) inhibitors. Our experimental approach is believed to be widely applicable for investigating the substrate specificity of other proteases in a rapid and high-throughput manner that is compatible for future automated analysis.

Amino Acid Substitution↗
PubMed2002

The role of amino-acid residues Q39 and E451 in the determination of substrate specificity of the Spodoptera frugiperda beta-glycosidase.

The design of beta-glycosidases with planed substrate specificity for biotechnological application has received little attention. This is mostly a consequence of the lack of data on the molecular basis of the beta-glycosidase specificity, namely data on the energy of the noncovalent interactions in the enzyme-transition state complex. In an attempt to fill this gap, site-directed mutagenesis and enzyme steady-state kinetic experiments with different substrates were conducted, using as model a digestive beta-glycosidase (glycoside hydrolase family 1) from Spodoptera frugiperda (Lepidoptera) (Sfbetagly50). The active site of this enzyme was modeled based on its sequence and on crystallographic data of similar enzymes. Energy of noncovalent interactions in transition state between Sfbetagly50 amino acids and glycone hydroxyls was determined. Sfbetagly50 residue E451 seems to be a key residue in determining beta-glycosidase preference for glucosides vs. galactosides based on the following data: (a) The energy of the noncovalent interaction between glycone equatorial hydroxyl 4 with E451 in the transition state is about 60% higher than its interaction with Q39. (b) The energy of the E451-hydroxyl 4 interaction decreases more than the Q39-hydroxyl 4 interaction when hydroxyl 4 is changed from equatorial to axial position. (c) A Sfbetagly50 mutant, where E451 was substituted by A, hydrolyzes galactosides faster than glucosides. It was also shown that glycone hydroxyl 6 interacts favorably with Q39, but not with E451, probably due to steric hindrance. These interactions result in the beta-glycosidase hydrolyzing fucosides (6-deoxygalactosides) faster than glucosides and galactosides.

Alanine↗
PubMed1999

Substrate-specific selenoprotein B of glycine reductase from Eubacterium acidaminophilum. Biochemical and molecular analysis.

The substrate-specific selenoprotein B of glycine reductase (PBglycine) from Eubacterium acidaminophilum was purified and characterized. The enzyme consisted of three different subunits with molecular masses of about 22 (alpha), 25 (beta) and 47 kDa (gamma), probably in an alpha 2 beta 2 gamma 2 composition. PBglycine purified from cells grown in the presence of [75Se]selenite was labeled in the 47-kDa subunit. The 22-kDa and 47-kDa subunits both reacted with fluorescein thiosemicarbazide, indicating the presence of a carbonyl compound. This carbonyl residue prevented N-terminal sequencing of the 22-kDa (alpha) subunit, but it could be removed for Edman degradation by incubation with o-phenylenediamine. A DNA fragment was isolated and sequenced which encoded beta and alpha subunits of PBglycine (grdE), followed by a gene encoding selenoprotein A (grdA2) and the gamma subunit of PBglycine (grdB2). The cloned DNA fragment represented a second GrdB-encoding gene slightly different from a previously identified partial grdBl-containing fragment. Both grdB genes contained an in-frame UGA codon which confirmed the observed selenium content of the 47-kDa (gamma) subunit. Peptide sequence analyses suggest that grdE encodes a proprotein which is cleaved into the previously sequenced N-terminal 25-kDa (beta) subunit and a 22-kDa (alpha) subunit of PBglycine. Cleavage most probably occurred at an -Asn-Cys- site concomitantly with the generation of the blocking carbonyl moiety from cysteine at the alpha subunit.

Amino Acid Oxidoreductases↗
PubMed1987

Substrate specificity of uridine and purine nucleoside phosphorylases of the whole cells of Escherichia coli.

Substrate specificity of uridine and purine nucleoside phosphorylases of the whole cells of Escherichia coli BM-11 has been studied. Both enzymes reveal similar requirements to the structure and stereochemistry of uracil nucleosides and of the pentofuranose-1-phosphates, respectively, viz, a) modifications at C-3' decreased the substrate activity to a greater extent as compared with the same modifications at C-2'; b) substitution of a methyl group for one of the 5'-CH2 protons does not lead to essential alterations of the substrate activity of such analogs vs. the natural substrates - uridine and ribofuranose-1-phosphate, respectively. PNP exhibits a very broad specificity for the purine acceptor.

Adenosine↗