Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “substrate specificity”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,279 records · Page 71Linked to original sources
PubMed1987

A new GTP-binding protein in brain tissues serving as the specific substrate of islet-activating protein, pertussis toxin.

A new GTP-binding protein serving as the specific substrate of islet-activating protein (IAP), pertussis toxin, was purified from porcine brain membranes as an alpha beta gamma-heterotrimeric structure. The alpha-subunit of the purified protein (alpha 40 beta gamma) had a molecular mass of 40 kDa and differed from that of Gi (alpha 41 beta gamma) or Go (alpha 39 beta gamma) previously purified from brain tissues. The fragmentation patterns of limited tryptic digestion and immunological cross-reactivities among the three alpha were different from one another. However, the beta gamma-subunit resolved from the three IAP substrates similarly inhibited a membrane-bound adenylate cyclase and their beta-subunits were immunologically indistinguishable from one another. Thus, the alpha 40 beta gamma is a new IAP substrate protein different from Gi or Go, in the alpha-subunit only.

Adenylate Cyclase Toxin↗
PubMed1995

Substrate-specificity of mammalian tissue-bound semicarbazide-sensitive amine oxidase.

Although the existence of a membrane-bound (probably plasmalemmal) semicarbazide-sensitive amine oxidase (SSAO) is well established in various mammalian tissues, and especially within vascular smooth muscle, its importance and the possible consequences of its metabolism of certain physiological and xenobiotic amines in vivo are under continuing investigation. In this respect, there are major species-related differences in substrate specificity determined in vitro, not only towards the synthetic amine benzylamine, but also towards some other aromatic amines (e.g. tyramine, tryptamine, 2-phenylethylamine, dopamine, histamine) which are possible endogenous substrates. Inhibition of SSAO can potentiate the pharmacological activity of some amines in isolated tissue (e.g. blood vessel) preparations from some species. Recent evidence has accumulated that SSAO may also be involved in metabolizing endogenous aliphatic amines such as methylamine and aminoacetone, focussing attention on the fact that the aldehyde products (formaldehyde and methylglyoxal, respectively) are potentially cytotoxic agents. Indeed, SSAO has been implicated in experimental models of cardiovascular toxicity involving conversion of the industrial aliphatic amine allylamine to acrolein. In summary, metabolism by SSAO may reduce the physiological/pharmacological effects of some amines, but the resulting metabolites (aldehydes, H2O2) may also have important actions.

Amine Oxidase (Copper-Containing)↗
PubMed2000

Application of a fluorescent histone acetyltransferase assay to probe the substrate specificity of the human p300/CBP-associated factor.

Histone N-acetyltransferases (HATs) are a group of enzymes which acetylate specific lysine residues in the N-terminal tails of nucleosomal histones to promote transcriptional activation. Recent structural and enzymatic work on the GCN5/PCAF HAT family has elucidated the structure of their catalytic domain and mechanism of histone acetylation. However, the substrate specificity of these enzymes has not been quantitatively investigated. Utilizing a novel microplate fluorescent HAT assay which detects the enzymatic production of coenzyme A (CoA), we have compared the activities of the HAT domains of human PCAF and its GCN5 homologue from yeast and Tetrahymena and found that they have similar kinetic parameters. PCAF was further assayed with a series of different length histone H3 peptide substrates, which revealed that the determinants for substrate recognition lie within a 19-residue sequence. Finally, we evaluated the acetylation of three putative PCAF substrates, histones H3 and H4 and the transcription factor p53, and have determined that histone H3 is significantly preferred over the histone H4 and p53 substrates. Taken together, the fluorescent acetyltransferase assay presented here should be widely applicable to other HAT enzymes, and the results obtained with PCAF demonstrate a strong substrate preference for the N-terminal residues of histone H3.

Acetyl Coenzyme A↗
PubMed1993

Substrate specificity characterization of a cdc2-like protein kinase purified from bovine brain.

A serine/threonine kinase from bovine brain has been purified (Lew, J., Beaudette, K. N., Litwin, C. M. E., and Wang, J. H. (1992) J. Biol. Chem. 267, 13383-13390) and found to consist of a 33-kDa catalytic subunit having high sequence homology to p34cdc2 and cdk2 (Lew, J., Winkfein, R. J., Paudel, H., and Wang, J. H. (1992) J. Biol. Chem. 267, 25922-25926). Substrate specificity determinants for this cdc2-like kinase were examined using synthetic peptide substrates derived from the in vitro p34cdc2 phosphorylation sites of histone H1. The peptide P-K-T-P-K-K-A-K-K-L was found to be an excellent substrate for the bovine cdc2-like kinase, having a Km value in the micromolar range. Important determinants for efficient substrate phosphorylation of this peptide were found both within the proposed substrate consensus motif (S/T-P-X-K/R) of p34cdc2 kinase and outside of this sequence. In addition to the absolute requirement for a proline residue immediately COOH-terminal to the phosphorylatable residue (+1) and a basic residue at the +3 position, a basic amino acid at the +2 position was greatly preferred over an acidic amino acid. A proline residue at the -2 position and a cluster of basic amino acids further COOH-terminal to the consensus motif were also found to be important for substrate binding. HeLa cell p34cdc2 kinase displays similar specificity to that of the bovine cdc2-like kinase, as the additional determinants outside of the consensus motif that contribute to the efficient phosphorylation of the histone peptide by this novel enzyme also appear to be important for p34cdc2-catalyzed phosphorylation.

Amino Acid Sequence↗
PubMed2004

Substrate specificity of Xenopus matrix metalloproteinase stromelysin-3.

The matrix metalloproteinase stromelysin-3 (ST3 or MMP11) was initially identified as a breast carcinoma associated protease and has since been shown to be highly expressed in diverse carcinomas and in developmental processes that involve extensive cell death (apoptosis) and tissue remodeling. Unlike other MMPs, purified ST3 has little activity toward known extracellular matrix (ECM) proteins in vitro but cleaves strongly a few non-ECM, extracellular proteins, including human alpha1-proteinase inhibitor (alpha1-PI). To investigate the possibility of alpha1-PI as a conserved physiological substrate for ST3 during vertebrate development, we analyzed the ability of Xenopus laevis ST3 catalytic domain to cleave frog alpha1-PI. Surprisingly, we found the ST3 failed to recognize the site in alpha1-PI equivalent to the major cleavage site in human alpha1-PI by mammalian ST3. Sequence and mutagenic analysis revealed that multiple substitutions at P2-P3' positions between human and Xenopus alpha1-PI contributed to the inability of Xenopus alpha1-PI to be cleaved by ST3. Our studies showed that (A)(G/A)(A)(M)(F/A)(L) (P3-P3') as a preferred cleavage site for ST3. We further demonstrated that mutations in the cleavage sites affected cleavage by ST3 differently from several other MMPs. These findings, together with earlier reports on ST3, showed that ST3 has distinct substrate specificities compared to other MMPs. Our results further suggest that alpha1-PI is unlikely to be a physiological substrate for ST3, at least with regard to evolutionarily conserved developmental processes.

Animals↗
PubMed1992

An engineered retroviral proteinase from myeloblastosis associated virus acquires pH dependence and substrate specificity of the HIV-1 proteinase.

In an attempt to understand the structural reasons for differences in specificity and activity of proteinases from two retroviruses encoded by human immunodeficiency virus (HIV) and myeloblastosis associated virus (MAV), we mutated five key residues predicted to form part of the enzyme subsites S1, S2 and S3 in the substrate binding cleft of the wild-type MAV proteinase wMAV PR. These were changed to the residues occupying a similar or identical position in the HIV-1 enzyme. The resultant mutated MAV proteinase (mMAV PR) exhibits increased enzymatic activity, altered substrate specificity, a substantially changed pH activity profile and a higher pH stability close to that observed in the HIV-1 PR. This dramatic alteration of MAV PR activity achieved by site-directed mutagenesis suggests that we have identified the amino acid residues contributing substantially to the differences between MAV and HIV-1 proteinases.

Amino Acid Sequence↗
PubMed2003

Studies on the substrate specificity of Escherichia coli galactokinase.

In vitro glycorandomization (IVG) technology is dependent upon the ability to rapidly synthesize sugar phosphates. Compared with chemical synthesis, enzymatic (kinase) routes to sugar phosphates would be attractive for this application. This work focuses upon the development of a high-throughput colorimetric galactokinase (GalK) assay and its application toward probing the substrate specificity and kinetic parameters of Escherichia coli GalK. The demonstrated dinitrosalicylic assay should also be generally applicable to a variety of sugar-processing enzymes. [reaction: see text]

Colorimetry↗
PubMed1982

Substrate specificity of the collagenolytic serine protease from Uca pugilator: studies with collagenous substrates.

The collagenolytic protease from Uca pugilator was studied with respect to its catalytic properties on collagen types I-V. The crab protease degraded all five collagen types, producing multiple cleavages in the triple helix of each native collagen at 25 degrees C. The major early cleavage in the alpha 1 polypeptide chain of collagen types I-III occurred at a 3/4:1/4 locus, resulting in fragments electrophoretically similar to the TCA and TCB products of mammalian collagenase action. Interestingly, a propensity toward this same cleavage was observed even following thermal denaturation of the substrates. The ability of the crab protease to degrade all native collagen types and to catalyze cleavages at multiple loci in the triple helix distinguishes its action from that of mammalian collagenases. The collagenolytic activity of the crab protease was also examined on fibrillar collagen and compared to that of human skin fibroblast collagenase. Enzyme concentrations of fibroblast collagenase which resulted in the saturation of available substrate sites failed to show such an effect in the case of the crab protease. Binding studies of the crab protease to fibrillar collagen likewise indicated substantially reduced levels of enzyme binding in comparison to fibroblast collagenase. These data suggest that the affinity of the crab protease for native collagen is considerably less than the affinity of mammalian collagenase for this substrate.

Animals↗
PubMed2006

Substrate specificity in enzymatic fluorination. The fluorinase from Streptomyces cattleya accepts 2'-deoxyadenosine substrates.

The fluorinase enzyme from Streptomyces cattleya displays an unusual ability in biocatalysis in that it forms a C-F bond. We now report that the enzyme will accept 2'-deoxyadenosine in place of adenosine substrates, and structural evidence reveals a reorganisation in hydrogen bonding to accommodate this substrate series. It emerges from this study that the enzyme does not require a planar ribose conformation of the substrate to catalyse C-F bond formation.

Bacterial Proteins↗
PubMed1980

On the substrate specificity of a damage-specific DNA binding protein from human cells.

The nature of the lesion recognized by a damage-specific DNA binding protein from human cells was investigated by examining the substrate specificity of this protein. Protein-recognizable damage was introduced into both T7 DNA and Poly d(A-T) at relatively low UV fluences. In addition, the protein demonstrated binding to both nitrous acid- and bisulfite-treated DNA, but not to DNA crosslinked with trioxsalen plus near-UV nor to non-irradiated uracil-containing DNA. These results suggest that this protein could be recognizing minor helix distortions in DNA rather than any one single lesion.

Carrier Proteins↗
PubMed1992

Substrate specificity of small-intestinal lactase. Assessment of the role of the substrate hydroxyl groups.

Lactase-phlorizin hydrolase is a disaccharidase present in the small intestine of mammals. This enzyme has two active sites, one being responsible for the hydrolysis of lactose. Lactase activity is thought to be selective towards glycosides with a hydrophilic aglycon. In this work, we report a systematic study on the importance of each hydroxyl group in the substrate molecule for lactase activity. For this purpose, all of the monodeoxy derivatives of methyl beta-lactoside and other lactose analogues are studied as lactase substrates. With respect to the galactose moiety, it is shown here that HO-3' and HO-2' are necessary for hydrolysis of the substrates by lactase. Using these chemically modified substrates, it has been confirmed that lactase does not behave as a typical beta-galactosidase, since it does not show an absolute selectivity with respect to substitution and stereochemistry at C4' in the galactose moiety of the substrate. However, the glucose moiety, in particular the HO-6, appears to be important for substrate hydrolysis, although none of the hydroxyl groups seemed to be essential. In order to differentiate both activities of the enzyme, a new assay for the phlorizin-hydrolase activity has also been developed.

Animals↗
PubMed1981

Characterization of 2-aminoisobutyric acid transport in Neurospora crassa: a general amino acid permease-specific substrate.

We report the characterization of an amino acid 2-aminoisobutyric acid was transported solely by the general amino acid permease and not by the neutral amino acid permease. Furthermore, this substrate was not metabolized after transport. The potential for a system-specific nonmetabolizable substrate as a tool in the analysis of amino acid transport and its regulation is discussed.

Amino Acid Transport Systems↗
PubMed1983

Substrate specificity of tissue plasminogen activator and urokinase as determined with synthetic chromogenic substrates.

Three different synthetic chromogenic substrates )H-glutamylglycyl-L-arginine-p-nitroanilide (S-2227), pyro-glutamyl-glycyl-L-arginine-p-nitroanilide (S-2444), and H-D-isoleucyl-L-prolyl-L-arginine-p-nitroanilide (S-2288] were investigated for use in the measurement of plasminogen activator activity with high molecular weight urokinase (H-UK), low molecular weight urokinase (L-UK), and tissue plasminogen activator (TPA). The three substrates were hydrolyzed by both TPA-type and UK-type plasminogen activator. As regards the amidolytic activity of S-2227, TPA exhibited a weaker amidolytic activity, and L-UK a stronger activity. In the case of the amidolytic activity of S-2444, no great difference between the three activators was observed in terms of Vmax. As regards the amidolytic activity of S-2288, L-UK exhibited a stronger activity, and TPA a weaker activity. It is suggested that the molecular size of the synthetic chromogenic substrate was too small when compared to natural substrate (fibrin), and therefore that fibrin-binding sites around the catalytic site in TPA are not recognized.

Chromogenic Compounds↗
PubMed2003

Ara12 subtilisin-like protease from Arabidopsis thaliana: purification, substrate specificity and tissue localization.

A C-terminal portion of Ara12 subtilisin-like protease (residues 542-757) was expressed in Escherichia coli cells as a fusion protein bound to maltose binding protein. Polyclonal antisera raised against the expressed protein were used to examine the tissue specificity and subcellular localization of Ara12. The protease was found predominantly in the silique and stem of plants, but was hardly detectable in leaf and not seen in root tissue. The distribution observed using immunological techniques is different from that seen by an RNA analysis study, which demonstrated similar mRNA abundance in the stem and leaves. Using immunogold labelling, Ara12 was shown to have an extracellular localization and was found in the intercellular spaces in stem tissue. Ara12 protease was purified to homogeneity from Arabidopsis thaliana cell suspension cultures by anion exchange and hydrophobic interaction chromatography. Proteolytic activity of Ara12 was inhibited by a number of serine protease inhibitors, but was almost unaffected by inhibitors of other catalytic classes of proteases. Optimal proteolytic activity was displayed under acidic conditions (pH 5.0). Ara12 activity was relatively thermostable and was stimulated in the presence of Ca2+ ions. Substrate specificity studies were conducted using a series of internally quenched fluorogenic peptide substrates. At the P1 position of substrates, hydrophobic residues, such as Phe and Ala, were preferred to Arg, whilst at the P1' position, Asp, Leu and Ala were most favoured. Possible functions of Ara12 are discussed in the light of the involvement of a number of plant subtilisin-like proteases in morphogenesis.

Arabidopsis↗
PubMed1995

On the substrate specificity of alpha-crystallin as a molecular chaperone.

alpha-Crystallin, the major protein of the ocular lens, acts as a molecular chaperone by preventing the thermal aggregation of proteins. However, in contrast with many other heat shock proteins, alpha-crystallin fails to protect proteins from aggregation during refolding reactions. Our results indicate that alpha-crystallin has substrate specificity different from other chaperones and recognizes specific non-native intermediates formed on the denaturation pathway only, with no affinity for intermediates formed on the refolding pathway.

Animals↗
PubMed2006

Substrate specificity of carboxylesterase isozymes and their contribution to hydrolase activity in human liver and small intestine.

Hydrolase activity from human liver and small intestine microsomes was compared with that of recombinant human carboxylesterases, hCE-1 and hCE-2. Although both hCE-1 and hCE-2 are present in human liver, the dominant component was found to be hCE-1, whereas the hydrolase activity of the human small intestine was found to be predominantly hCE-2. hCE-2 has a limited ability to hydrolyze large acyl compound substrates. Interestingly, propranolol derivatives, good substrates for hCE-2, were easily hydrolyzed by substitution of the methyl group on the 2-position of the acyl moiety, but were barely hydrolyzed when the methyl group was substituted on the 3-position. These findings suggest that hCE-2 does not easily form acylated intermediates because of conformational interference in its active site. In contrast, hCE-1 could hydrolyze a variety of substrates. The hydrolytic activity of hCE-2 increased with increasing alcohol chain length in benzoic acid derivative substrates, whereas hCE-1 preferentially catalyzed the hydrolysis of substrates with short alcohol chains. Kinetic data showed that the determining factor for the rate of hydrolysis of p-aminobenzoic acid esters was V(max) for hCE-1 and K(m) for hCE-2. Furthermore, the addition of hydrophobic alcohols to the reaction mixture with p-aminobenzoic acid propyl ester induced high and low levels of transesterification by hCE-1 and hCE-2, respectively. When considering the substrate specificities of hCE-1, it is necessary to consider the transesterification ability of hCE-1, in addition to the binding structure of the substrate in the active site of the enzyme.

Animals↗
PubMed1979

Modification of the substrate specificity of rat hepatic lipase by collagenase treatment.

Collagenase is currently used in the isolation of rat hepatocytes, but it rapidly inactivates the heparin-releasable triacylglycerol lipase of the liver. Since collagenase-isolated liver cells contain a heparin-releasable monoacylglycerol hydrolase, a study was made on the effect of collagenase treatment on the substrate specificity of purified heparin-releasable lipase of rat liver. Incubation of the purified lipase with collagenase selectively decreased the triacylglycerol lipase activity of the enzyme with no effect on the monoacylglycerol hydrolase activity. Gel filtration of the lipase before and after collagenase treatment indicated cleavage of a small molecular weight fragment from the enzyme. This resulted in a preparation with less triacylglycerol lipase activity but still capable of monoacylglycerol hydrolysis.

Animals↗
PubMed2004

Crystal structures of Escherichia coli uridine phosphorylase in two native and three complexed forms reveal basis of substrate specificity, induced conformational changes and influence of potassium.

Uridine phosphorylase (UP) is a key enzyme in the pyrimidine salvage pathway that catalyses the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate. Inhibiting liver UP in humans raises blood uridine levels and produces a protective effect ("uridine rescue") against the toxicity of the chemotherapeutic agent 5-fluorouracil without reducing its antitumour activity. We have investigated UP-substrate interactions by determining the crystal structures of native Escherichia coli UP (two forms), and complexes with 5-fluorouracil/ribose 1-phosphate, 2-deoxyuridine/phosphate and thymidine/phosphate. These hexameric structures confirm the overall structural similarity of UP to E.coli purine nucleoside phosphorylase (PNP) whereby, in the presence of substrate, each displays a closed conformation resulting from a concerted movement that closes the active site cleft. However, in contrast to PNP where helix segmentation is the major conformational change between the open and closed forms, in UP more extensive changes are observed. In particular a swinging movement of a flap region consisting of residues 224-234 seals the active site. This overall change in conformation results in compression of the active site cleft. Gln166 and Arg168, part of an inserted segment not seen in PNP, are key residues in the uracil binding pocket and together with a tightly bound water molecule are seen to be involved in the substrate specificity of UP. Enzyme activity shows a twofold dependence on potassium ion concentration. The presence of a potassium ion at the monomer/monomer interface induces some local rearrangement, which results in dimer stabilisation. The conservation of key residues and interactions with substrate in the phosphate and ribose binding pockets suggest that ribooxocarbenium ion formation during catalysis of UP may be similar to that proposed for E.coli PNP.

Catalytic Domain↗