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PubMed1981

Substrate specificity and histochemical distribution of aminopeptidase in rat testis and epididymis.

The substrate specificity of rat testicular and epididymal peptidase was investigated using chromogenic substrates, D. L-alanine-, L-arginine-, gamma-N-L-glutamine-, L-leucine-, D. L-methionine-, alpha-N-benzoyl-D. L-arginine-, and N-benzoyl-L-leucine-beta-naphthylamide. The histochemical distribution of peptidase activity demonstrated with these substrates was also investigated in the testis and epididymis. L-Arginine-beta-raphthylamide (Arg-beta-NA) and gamma-N-L-glutamine-beta-naphthylamide (Glu-beta-NA) were mostly hydrolyzed in the testis and epididymis, respectively. Histologically, the activity using Arg-beta-NA as substrate (aminopeptidase B) appeared in both the cytoplasms and nuclei of interstitial cells and spermatogonia and the heads of spermatozoa, while activity using other substrates was found only in the cytoplasms of cells in the germinal epithelium. In the epididymis, strong reaction with Glu-beta-NA (gamma-glutamyl transpeptidase) was found in the apical part of the epithelial cells and in the heads of spermatozoa. Neither alpha-N-benzoyl-L-arginine- nor N-benzoyl-L-leucine-beta-naphthylamide was utilized in either the testis or the epididymis.

Aminopeptidases↗
PubMed2002

The substrate specificity of tripartite efflux systems of Pseudomonas aeruginosa is determined by the RND component.

The tripartite efflux systems MexAB-OprM and MexCD-OprJ of Pseudomonas aeruginosa each display characteristic substrate specificity against a variety of antimicrobial agents. The chimeric efflux system MexC-MexB-OprJ/DeltaMexD constructed by exchange of MexD with MexB endowed the recombinant host the same resistance profile as MexAB-OprM rather than MexCD-OprJ. The change of substrate specificity was shown to be due to extrusion from the chimeric efflux system by cellular accumulation experiments using tetracycline, erythromycin, and ethidium bromide. Thus, we conclude that MexB and MexD are primary components of the efflux system responsible for sorting extrusion substrates.

Anti-Bacterial Agents↗
PubMed2002

The physiological role of RNase T can be explained by its unusual substrate specificity.

Escherichia coli RNase T, the enzyme responsible for the end-turnover of tRNA and for the 3' maturation of 5 S and 23 S rRNAs and many other small, stable RNAs, was examined in detail with respect to its substrate specificity. The enzyme was found to be a single-strand-specific exoribonuclease that acts in the 3' to 5' direction in a non-processive manner. However, although other Escherichia coli exoribonucleases stop several nucleotides downstream of an RNA duplex, RNase T can digest RNA up to the first base pair. The presence of a free 3'-hydroxyl group is required for the enzyme to initiate digestion. Studies with RNA homopolymers and a variety of oligoribonucleotides revealed that RNase T displays an unusual base specificity, discriminating against pyrimidine and, particularly, C residues. Although RNase T appears to bind up to 10 nucleotides in its active site, its specificity is defined largely by the last 4 residues. A single 3'-terminal C residue can reduce RNase T action by >100-fold, and 2-terminal C residues essentially stop the enzyme. In vivo, the substrates of RNase T are similar in that they all contain a double-stranded stem followed by a single-stranded 3' overhang; yet, the action of RNase T on these substrates differs. The substrate specificity described here helps to explain why the different substrates yield different products, and why certain RNA molecules are not substrates at all.

Base Sequence↗
PubMed2003

Probing protein-tyrosine phosphatase substrate specificity using a phosphotyrosine-containing phage library.

Protein tyrosine phosphatases (PTPs) play important, highly dynamic roles in signaling. Currently about 90 different PTP genes have been described. The enzymes are highly regulated at all levels of expression, and it is becoming increasingly clear that substrate specificity of the PTP catalytic domains proper contributes considerably to PTP functionality. To investigate PTP substrate selectivity, we have set up a procedure to generate phage libraries that presents randomized, phosphotyrosine-containing peptides. Phages that expressed suitable substrates were selected by immobilized, substrate-trapping GST-PTP fusion proteins. After multiple rounds of selection, positive clones were confirmed by SPOT analysis, dephosphorylation by wild-type enzyme, and Km determinations. We have identified distinct consensus substrate motifs for PTP1B, Sap-1, PTP-beta, SHP1, and SHP2. Our results confirm substrate specificity for individual PTPs at the peptide level. Such consensus sequences may be useful both for identifying potential PTP substrates and for the development of peptidomimetic inhibitors.

Amino Acid Sequence↗
PubMed2004

Analysis of the substrate specificity of the Staphylococcus aureus sortase transpeptidase SrtA.

The Staphylococcus aureus sortase transpeptidase SrtA isoform is responsible for the covalent attachment of virulence and colonization-associated proteins to the bacterial peptidoglycan. SrtA utilizes two substrates, undecaprenol-pyrophosphoryl-MurNAc(GlcNAc)-Ala-D-isoGlu-Lys(epsilon-Gly(5))-D-Ala-D-Ala (branched Lipid II) and secreted proteins containing a highly conserved C-terminal LPXTG sequence. SrtA simultaneously cleaves the Thr-Gly bond of the LPXTG-containing protein and forms a new amide bond with the nucleophilic amino group of the Gly(5) portion of branched Lipid II, anchoring the protein to this key intermediate that is subsequently polymerized into peptidoglycan. Here we describe the development of a general in vitro method for elucidating the substrate specificity of sortase enzymes. In addition, using immunofluorescence, cell adhesion assays, and transmission electron microscopy, we establish links between in vitro substrate specificity and in vivo function of the S. aureus sortase isoforms. Results from these studies provide strong supporting evidence of a primary role of the SrtA isoform in S. aureus adhesion and host colonization, illustrate a lack of specificity cross talk between SrtA and SrtB isoforms, and highlight the potential of SrtA as a target for the development of antivirulence chemotherapeutics against Gram-positive bacterial pathogens.

Amino Acid Motifs↗
PubMed1998

Roles of amino acids 161 to 179 in the PSE-4 omega loop in substrate specificity and in resistance to ceftazidime.

The PSE-4 enzyme is a prototype carbenicillin-hydrolyzing enzyme exhibiting high activity against penicillins and early cephalosporins. To understand the mechanism that modulates substrate profiles and to verify the ability of PSE-4 to extend its substrate specificity toward expanded-spectrum cephalosporins, we used random replacement mutagenesis to generate six random libraries from amino acids 162 to 179 in the Omega loop. This region is known from studies with TEM-1 to be implicated in substrate specificity. It was found that the mechanism modulating ceftazidime hydrolysis in PSE-4 was different from that in TEM-1. The specificity of class 2c carbenicillin-hydrolyzing enzymes could not be assigned to the Omega loop of PSE-4. Analysis of the percentage of functional enzymes revealed that the hydrolysis of ampicillin was more affected than hydrolysis of carbenicillin by amino acid substitutions at positions 162 to 164 and 165 to 167.

Amino Acid Sequence↗
PubMed1997

Structure of human isovaleryl-CoA dehydrogenase at 2.6 A resolution: structural basis for substrate specificity,.

Isovaleryl-CoA dehydrogenase (IVD) belongs to an important flavoprotein family of acyl-CoA dehydrogenases that catalyze the alpha,beta-dehydrogenation of their various thioester substrates. Although enzymes from this family share similar sequences, catalytic mechanisms, and structural properties, the position of the catalytic base in the primary sequence is not conserved. E376 has been confirmed to be the catalytic base in medium-chain (MCAD) and short-chain acyl-CoA dehydrogenases and is conserved in all members of the acyl-CoA dehydrogenase family except for IVD and long-chain acyl-CoA dehydrogenase. To understand this dichotomy and to gain a better understanding of the factors important in determining substrate specificity in this enzyme family, the three-dimensional structure of human IVD has been determined. Human IVD expressed in Escherichia coli crystallizes in the orthorhombic space group P212121 with unit cell parameters a = 94.0 A, b = 97.7 A, and c = 181.7 A. The structure of IVD was solved at 2.6 A resolution by the molecular replacement method and was refined to an R-factor of 20.7% with an Rfree of 28.8%. The overall polypeptide fold of IVD is similar to that of other members of this family for which structural data are available. The tightly bound ligand found in the active site of the structure of IVD is consistent with that of CoA persulfide. The identity of the catalytic base was confirmed to be E254, in agreement with previous molecular modeling and mutagenesis studies. The location of the catalytic residue together with a glycine at position 374, which is a tyrosine in all other members of the acyl-CoA dehydrogenase family, is important for conferring branched-chain substrate specificity to IVD.

Acyl Coenzyme A↗
PubMedDate not supplied

[Activity and substrate specificity of the alcohol dehydrogenases of n-alkane oxidizing yeasts].

The activity and substrate specificity of alcohol dehydrogenases (ADH) in the fractions of cytosol and membrane particles were compared in the yeasts Torulopsis candida, Candida lipolytica and Candida tropicalis grown in media with glucose and hexadecane. In all studied yeast cultures growing in the medium with hexadecane, NAD-dependent ADH specifically dehydrogenating only medium and higher alcohols are induced in the membrane structures of the cells. Soluble ADH are found in the cytosol of the cultures grown either on glucose or on hexadecane. These ADH oxidize all alcohols with the carbon chain length from C2 to C16. As was found by electrophoresis in polyacrylamide gel, the number of ADH molecular forms in the cytosol fraction of the cultures depends on the carbon growth substrate being used and the peculiarities of yeast culture.

Alcohol Oxidoreductases↗
PubMed2002

Substrate specificity of schistosome versus human legumain determined by P1-P3 peptide libraries.

Asparaginyl endopeptidases, or 'legumains' have been identified and characterized in plants, the blood fluke parasite Schistosoma, and mammals. The legumains are a novel family of cysteine proteases and display restricted specificity for peptide hydrolysis on the carboxyl side of asparagine residues. Two forms of recombinant asparaginyl endopeptidase from Schistosoma mansoni (C197 Sm32 and N197C Sm32), expressed in Pichia pastoris, have been analyzed for substrate specificity using a positional-scanning synthetic combinatorial library (PS-SCL). We first screened Sm32 using a P1-diverse library. This library demonstrated the absolute specificity of Sm32 for asparagine at P1. To determine the P2-P3 preferences of Sm32, we constructed a library with asparagine fixed at P1, and the P2-P3 positions randomized. The library was screened using the two forms of Sm32, human asparaginyl endopeptidase, and to confirm its diversity, cruzain from Trypanosoma cruzi. The schistosome legumain showed a preference for P3: Thr>Ala>Val>Ile, and P2: Ala>Thr>Val>Asn, with an overall broader specificity at P3 than at P2. Both human and schistosome legumain can accommodate Thr and Ala at P2 and P3. However, optimal substrate sequences differ, with Sm32 preferring Thr-Ala-Asn, and human legumain preferring Pro-Thr-Asn. Predictions of substrate specificity from the library screen were confirmed using single peptide substrates for kinetic assays.

Amino Acid Sequence↗
PubMed1987

Recruitment of substrate-specificity properties from one enzyme into a related one by protein engineering.

The Bacillus licheniformis and Bacillus amyloliquefaciens subtilisins differ by 31% in protein sequence and by factors of greater than 60 in catalytic efficiency, kcat/Km, toward various substrates. Despite large differences in sequence and substrate specificity for these serine proteases, only two amino acid substitutions (residues 156 and 217) occur within 4 A (contact distance) of modeled substrates, and a third substitution (residue 169) is within 7 A. The three B. licheniformis substitutions (Ser-156/Ala-169/Leu-217) were introduced into the wild-type B. amyloliquefaciens subtilisin (Glu-156/Gly-169/Tyr-217) by site-directed mutagenesis. The substrate specificity of the triple mutant approaches that of B. licheniformis enzyme when assayed with seven different substrates that vary in charge, size, and hydrophobicity. Thus, specificity properties of distantly related and functionally divergent enzymes can be exchanged by limited amino acid replacements, in this case representing less than 4% of the sequence differences.

Amino Acids↗
PubMed1991

A single amino acid substitution changes the substrate specificity of quinoprotein glucose dehydrogenase in Gluconobacter oxydans.

Gluconobacter oxydans contains pyrroloquinoline quinone-dependent glucose dehydrogenase (GDH). Two isogenic G. oxydans strains, P1 and P2, which differ in their substrate specificity with respect to oxidation of sugars have been analysed. P1 can oxidize only D-glucose, whereas P2 is also capable of the oxidation of the disaccharide maltose. To investigate the nature of this maltose-oxidizing property we cloned the gene encoding GDH from P2. Expression of P2 gdh in P1 enables the latter strain to oxidize maltose, indicating that a mutation in the P2 gdh gene is responsible for the change in substrate specificity. This mutation could be ascribed to a 1 bp substitution resulting in the replacement of His 787 by Asn.

Amino Acid Sequence↗
PubMed1993

Substrate specificity of 'elastomucoproteinase': an enzyme which can degrade cartilage aggrecan.

The substrate specificity of elastomucoproteinase (EMP), an enzyme which was first isolated from crude pancreatic elastase and described as a proteoglycan-degrading enzyme, determined on tripeptide-p-nitroanilide substrates indicates the existence of a 'new' chymotrypsin-like enzyme. EMP, however, did not cleave any glycosaminoglycans, i.e., its 'mucolytic' effect has been excluded. Activity of EMP on synthetic or protein substrates (e.g., collagen type-II and aggrecan of cartilage) was completely inhibited by serine proteinase inhibitors, which was also found when using cartilage proteoglycan monomers. EMP cleaves the core protein of proteoglycan monomer (aggrecan) into small peptides, some containing glycosaminoglycan chains resulting in an unusual elution profile on Sepharose CL-6B chromatography when compared to the effects of pancreatic and granulocyte elastases, chymotrypsin, cathepsin G and stromelysin. EMP-like activity also was detected in neutrophil granules of bovine leukocytes and polyclonal antibodies were raised against purified bovine EMP to detect the enzyme in both crude elastase preparations and the granule fraction of bovine leukocytes.

Adult↗
PubMed2002

Role of tryptophan hydroxylase phe313 in determining substrate specificity.

The active site residue phenylalanine 313 is conserved in the sequences of all known tryptophan hydroxylases. The tryptophan hydroxylase F313W mutant protein no longer shows a preference for tryptophan over phenylalanine as a substrate, consistent with a role of this residue in substrate specificity. A tryptophan residue occupies the homologous position in tyrosine hydroxylase. The tyrosine hydroxylase W372F mutant enzyme does not show an increased preference for tryptophan over tyrosine or phenylalanine, so that this residue cannot be considered the dominant factor in substrate specificity in this family of enzymes.

Animals↗
PubMed1977

Substrate specificity of antinuclear antibodies in scleroderma.

Studies of antinuclear antibodies (ANA) were carried out in 39 cases of systemic scleroderma and for comparison in 19 cases of systemic lupus erythematosus (SLE) and 4 of mixed connective tissue disease (MCTD) using indirect immunofluorescence (IF) methods under standard conditions. The results on three different substrates--monkey esophagus, guineapig lip and rat liver--are reported. In 48.7% of scleroderma cases ANA showed a substrate specificity. The highest percentage of positive results in scleroderma was obtained on monkey esophagus (97.4%) and the lowest on rat liver (61.5%). In SLE and MCTD, in contrast, only about 13% of the sera displayed such specificity. If only sera with substrate specificity are considered, the positive results on monkey esophagus and rat liver are 94.7% and 21.1%, respectively. Titers of sera reacting positively on 2 or 3 substrates were mostly in agreement, although some sera both in systemic scleroderma and SLE showed higher titers on monkey esophagus. The IF pattern was usually the same regardless of the substrate, Tests for ANA in scleroderma should be performed on at least 2 substrates simultaneously.

Adult↗
PubMed1998

Substrate specificity of the rat liver Na(+)-bile salt cotransporter in Xenopus laevis oocytes and in CHO cells.

It has been proposed that the hepatocellular Na(+)-dependent bile salt uptake system exhibits a broad substrate specificity in intact hepatocytes. In contrast, recent expression studies in mammalian cell lines have suggested that the cloned rat liver Na(+)-taurocholate cotransporting polypeptide (Ntcp) may transport only taurocholate. To characterize its substrate specificity Ntcp was stably transfected into Chinese hamster ovary (CHO) cells. These cells exhibited saturable Na(+)-dependent uptake of [3H]taurocholate [Michaelis constant (K(m)) of approximately 34 microM] that was strongly inhibited by all major bile salts, estrone 3-sulfate, bumetanide, and cyclosporin A. Ntcp cRNA-injected Xenopus laevis oocytes and the transfected CHO cells exhibited saturable Na(+)-dependent uptake of [3H]taurochenodeoxycholate (Km of approximately 5 microM), [3H]tauroursodeoxycholate (Km of approximately 14 microM), and [14C]glycocholate (Km of approximately 27 microM). After induction of gene expression by sodium butyrate, Na(+)-dependent transport of [3H]estrone 3-sulfate (Km of approximately 27 microM) could also be detected in the transfected CHO cells. However, there was no detectable Na(+)-dependent uptake of [3H]bumetanide or [3H]cyclosporin A. These results show that the cloned Ntcp can mediate Na(+)-dependent uptake of all physiological bile salts as well as of the steroid conjugate estrone 3-sulfate. Hence, Ntcp is a multispecific transporter with preference for bile salts and other anionic steroidal compounds.

Animals↗
PubMed2004

Quantitative structure-activity relationship for the cleavage of C3/C4-substituted catechols by a prototypal extradiol catechol dioxygenase with broad substrate specificity.

Catechol 2,3-dioxygenase [EC 1.13.11.2] from Pseudomonas putida mt-2 (Mpc) catalyzes the extradiol cleavage of catechol to produce 2-hydroxymuconate semialdehyde. The K(m) values for the catecholic substrate (K(mA)) and O(2) (K(mO2)), and catalytic constants (k(cat)) were kinetically determined for eight C3/C4-substituted catechols at 25 degrees C and pH 6.5 or 7.5. The first pK(a) values (pK(1)) were determined for eleven catechols (pK(1) = 7.26-9.47), correlated with Hammett substituent constants, and electron-withdrawing substituents significantly stabilized the monoanionic species of free catechols. Mpc preferred catechols with non-ionic substituents at the C3 or C4 position. 3-Phenylcatechol, a biphenyl, was cleaved, while 4-tert-butylcatechol was not. The logarithm of k(cat)/K(mA) (substrate specificity constant) exhibited a good linear correlation with pK(1), with the exception of those for 4-halocatechols. The logarithm of k(cat)/K(mO2) showed a good linear correlation with pK(1), with the exception of that of 3-phenylcatechol. These results demonstrate that catechol binding to the Mpc active site, the following O(2) binding, and the activation of the bound O(2) are all sensitive to electronic effects of the substituents. However, k(cat) did not correlate significantly with pK(1). The present study distinguishes clearly between the electronic and the steric effects of catecholic substrates in the reactivity of Mpc, and provides important insight into the mechanistic basis for a vast range of substrate specificities of extradiol dioxygenases.

Binding Sites↗
PubMed2003

Computational approach for prediction of domain organization and substrate specificity of modular polyketide synthases.

Modular polyketide synthases (PKSs) are large multi-enzymatic, multi-domain megasynthases, which are involved in the biosynthesis of a class of pharmaceutically important natural products, namely polyketides. These enzymes harbor a set of repetitive active sites termed modules and the domains present in each module dictate the chemical moiety that would add to a growing polyketide chain. This modular logic of biosynthesis has been exploited with reasonable success to produce several novel compounds by genetic manipulation. However, for harnessing their vast potential of combinatorial biosynthesis, it is essential to develop knowledge based in silico approaches for correlating the sequence and domain organization of PKSs to their polyketide products. In this work, we have carried out extensive sequence analysis of experimentally characterized PKS clusters to develop an automated computational protocol for unambiguous identification of various PKS domains in a polypeptide sequence. A structure based approach has been used to identify the putative active site residues of acyltransferase (AT) domains, which control the specificities for various starter and extender units during polyketide biosynthesis. On the basis of the analysis of the active site residues and molecular modelling of substrates in the active site of representative AT domains, we have identified a crucial residue that is likely to play a major role in discriminating between malonate and methylmalonate during selection of extender groups by this domain. Structural modelling has also explained the experimentally observed chiral preference of AT domain in substrate selection. This computational protocol has been used to predict the domain organization and substrate specificity for PKS clusters from various microbial genomes. The results of our analysis as well as the computational tools for prediction of domain organization and substrate specificity have been organized in the form of a searchable computerized database (PKSDB). PKSDB would serve as a valuable tool for identification of polyketide products biosynthesized by uncharacterized PKS clusters. This database can also provide guidelines for rational design of experiments to engineer novel polyketides.

Amino Acid Sequence↗