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PubMed1998

Substrate specificity of delta ribozyme cleavage.

The specificity of delta ribozyme cleavage was investigated using a trans-acting antigenomic delta ribozyme. Under single turnover conditions, the wild type ribozyme cleaved the 11-mer ribonucleotide substrate with a rate constant of 0.34 min-1, an apparent Km of 17.9 nM and an apparent second-order rate constant of 1.89 x 10(7) min-1 M-1. The substrate specificity of the delta ribozyme was thoroughly investigated using a collection of substrates that varied in either the length or the nucleotide sequence of their P1 stems. We observed that not only is the base pairing of the substrate and the ribozyme important to cleavage activity, but also both the identity and the combination of the nucleotide sequence in the substrates are essential for cleavage activity. We show that the nucleotides in the middle of the P1 stem are essential for substrate binding and subsequent steps in the cleavage pathway. The introduction of any mismatches at these positions resulted in a complete lack of cleavage by the wild type ribozyme. Our findings suggest that factors more complex than simple base pairing interactions, such as tertiary structure interactions, could play an important role in the substrate specificity of delta ribozyme cleavage.

Base Sequence↗
PubMed1995

Molecular evolution of a class C beta-lactamase extending its substrate specificity.

Enterobacter cloacae GC1, a clinical strain isolated in 1992 in Japan, was found to produce a chromosomal class C beta-lactamase with extended substrate specificity to oxyimino beta-lactam antibiotics, significantly differing from the known E. cloacae beta-lactamases such as the P99 beta-lactamase. The 1560 nucleotides including the GC1 beta-lactamase gene were sequenced, and the amino acid sequence of the mature enzyme comprising 364 amino acids was deduced. A comparison of the amino acid sequence with those of known E. cloacae beta-lactamases revealed the duplication of three amino acids at positions 208-213, i.e. Ala-Val-Arg-Ala-Val-Arg. This duplication was attributed to a tandem duplication of a 9-nucleotide sequence. The chimeric beta-lactamases produced by the chimeric genes from the GC1 and P99 beta-lactamase genes indicated that the extended substrate specificity is entirely attributed to the 3-amino acid insertion. Two mutant beta-lactamases were prepared from P99 beta-lactamase by site-directed mutagenesis, i.e. an Ala-Ala-Ala sequence was inserted before or after the native Ala-Val-Arg at positions 208-210. These mutant enzymes revealed that the Ala-Val-Arg located from positions 211 to 213 in the GC1 beta-lactamase are the newly inserted residues, and this phenomenon is independent of the characteristics of the amino acids inserted.

Amino Acid Sequence↗
PubMed1995

Functional linkage between the active site of alpha-lytic protease and distant regions of structure: scanning alanine mutagenesis of a surface loop affects activity and substrate specificity.

Previous structural and kinetic characterization of mutations within the active site of alpha-lytic protease have demonstrated that amino acid residues in direct contact with the substrate are major substrate specificity determinants. The experiments described here identify residues 216-226 of alpha-lytic protease as a region of structure peripheral to the active site that also plays an important role in establishing the substrate specificity of the enzyme. Alanine substitution mutations within this surface loop of 19 amino acid residues significantly perturb the enzyme's specificity profile, despite being as far as 21 A from the hydroxyl group of Ser195. The kinetic consequences of the mutations are remarkably independent of position within the loop and suggest that active site plasticity is affected more than static structure. Kinetic characterization of double mutants with the Met190-->Ala broad-specificity active site mutation reveals varying degrees of non-additivity and indicates that active site plasticity can be influenced through multiple sets of interactions. Although these results clearly demonstrate that tuning of serine protease activity is possible through remodelling of structure surrounding the active site, practical issues such as retaining compatibility with the folding mechanism and stability of the mature enzyme present significant obstacles to general application of the technique.

Alanine↗
PubMed1994

Substrate specificity of collagenolytic proteases from the king crab Paralithodes camtschatica.

Substrate specificity of two collagenolytic proteases from the king crab Paralithodes camtschatica has been studied. Both proteases are shown to hydrolyze effectively type I and III collagens, gelatin and fibrinogen. The variety of products formed during the enzymatic hydrolysis of the proteins appeared to be different for crab proteases A and C. Studies on peptide hydrolysis demonstrated that protease A cleaves preferably peptide bonds with Arg and Lys as carbonyl components, while protease C prefers hydrophobic amino acids. Kinetic constants of hydrolysis for low molecular weight substrates in the presence of crab proteases have been determined. This allowed us to characterize collagenolytic protease A as a trypsin-like protease. By contrast, collagenolytic protease C was classified as chymotrypsin-like protease although this protease and bovine chymotrypsin are not completely similar. Collagenase substrates Pz-Pro-Leu-Gly-Pro-D-Arg and Z-Gly-Pro-Ala-Gly-Pro-Ala were found to be resistant to both crab proteases.

Amino Acid Sequence↗
PubMed1996

Substrate specificity of monomeric and dimeric alpha-sarcin.

The substrate specificity of monomeric and dimeric forms of alpha-sarcin was investigated by membrane blotting procedures. Dimeric alpha-sarcin fails to inactivate ribosomes as well as to hydrolyze mini-stem-loop RNA, whereas monomeric alpha-sarcin catalyzes both substrates. Both monomeric and dimeric alpha-sarcin are effective ribonucleases that are displayed by in situ RNA-impregnated gel electrophoresis. The same purine base specificity was detected for both dimeric and monomeric forms. alpha-Sarcin is also an effective deoxyribonuclease to supercoiled DNA. The action of alpha-sarcin as deoxyribonuclease and ribonuclease is inhibited by the presence of SDS (3.5 x 10(-6) M); the inhibition on ribonuclease, but not on deoxyribonuclease, is reversible if the proteins are renatured.

Animals↗
PubMed1998

Substrate specificity of pepstatin-insensitive carboxyl proteinase from Bacillus coagulans J-4.

Bacillus coagulans J-4 carboxyl proteinase, designated as J-4, is characterized as alcohol-resistant and insensitive to aspartic proteinase inhibitors such as pepstatin, diazoacetyl-DL-norleucinemetylester, and 1,2-epoxy-3-(p-nitrophenoxy)propane. Here, its substrate specificity was elucidated by using two series of chromogenic substrates, Lys-Pro-Ala-Lys-Phe*Nph (p-nitrophenylalanine:* is cleavage site)-Arg-Leu (XVI) and Lys-Pro-Ile-Glu-Phe*Nph-Arg-Leu (RS6), in which the amino acid residues at positions P5-P2, P2', and P3' were systematically substituted. Kinetic parameters were determined for both sets of peptides. J-4 was shown to hydrolyze Lys-Pro-Ala-Ala-Phe-Nph-Arg-Leu most effectively among the XVI series. The kinetic parameters of this peptide were Km = 20.0 +/- 3.24 microM, kcat = 15.4 +/- 0.71 s-1, and kcat/Km = 0.769 +/- 0.128 microM-1.s-1. Among the RS6 series, Lys-Pro-Ile-Pro-Phe-Nph-Arg-Leu was hydrolyzed most effectively. The kinetic parameters of this peptide were Km = 13.7 +/- 1.30 microM, kcat = 9.65 +/- 0.38 s-1, and kcat/Km = 0.704 +/- 0.072 microM-1.s-1. These systematic analyses revealed that J-4 had a unique preference for the P2 position: J-4 preferentially hydrolyzed peptides having an Ala or Pro residue in the P2 position. Other carboxyl proteinases preferred peptides having hydrophobic and bulky amino acid residue such as Leu in the P2 position. Thus, J-4 was found to differ considerably in substrate specificity from the other carboxyl proteinases reported so far.

Amino Acid Sequence↗
PubMed2004

Affinity chromatography of porcine pepsin and pepsinogen using immobilized ligands derived from the specific substrate for this enzyme.

Affinity chromatography of porcine protease and its zymogen was carried out on immobilized components of specific substrate used for the pepsin determination. For the immobilization of N-acetyl-L-phenylalanine and iodinated derivative of L-tyrosine, divinyl sulfone activated Sepharose was used. Ligands with blocked amino group and free carboxyl one were linked to Sepharose via ethylene diamine spacer using carbodiimide reaction. Conditions of affinity chromatography of porcine pepsin and pepsinogen on the prepared carriers were optimized: the effect of pH, ionic strength and a nature of the buffers used on adsorption of the enzyme and zymogen to an affinity carrier, as well as their elution was studied. The following parameters were taken into consideration: capacity of the prepared affinity matrices, reproducibility of experiments and the enzyme stability. Pepsin was adsorbed to both immobilized ligands at pH 3.5-4.0; for the elution of the enzyme it was necessary to increase ionic strength (up to 0.5 M). For the adsorption of pepsinogen pH 5.2 was found to be optimum, for its desorption, an increase of ionic strength was used.

Amino Acids↗
PubMed1978

2-deoxygalactose, a specific substrate of the Salmonella typhiimurium galactose permease: its use for the isolation of galP mutants.

2-Deoxygalactose is a specific substrate of the galactose permease. The apparent Km is about 500 micron, compared to 45 micron for galactose, whereas the maximal rate of uptake is one-half to one-third of that of galactose. None of the other galactose transport systems, including methyl beta-D-thiogalactosides I and II, the beta-methyl-galactoside permease, and both arabinose systems, is able to catalyze transport of 2-deoxygalactose to a significant extent. 2-Deoxygalactose can also be used to isolate mutants defective in galactose permease, since it is bacteriostatic. Colonies that grow with lactate, malate, or succinate as a carbon source in the presence of 0.5 to 2 mM 2-doexygalactose were found to be mostly galP mutants, lacking galactose permease. Spontaneous 2-deoxygalactose-resistant strains arose with a frequency of about 2 X 10(-6). galP mutants have also been derived from pts deletion mutants that require galactose permease for growth on glucose. Revertants have been obtained that have acquired the parental phenotype.

Biological Transport↗
PubMed1983

L-Pyroglutamyl-L-prolyl-L-valine-p-nitroanilide, a highly specific substrate for granulocyte elastase.

L-Pyroglutamyl-L-prolyl-L-valine-p-nitroanilide was found to be a highly specific substrate for human granulocyte elastase. At pH 8.3 and 37 degrees C, its Km = 0.55 mmol/l and the value for kcat was 6 sec-1, whereas with porcine pancreatic elastase these values were approximately 2 mmol/l and less than 0.001 sec-1, respectively. It is not cleaved by trypsin or chymotrypsin. With granulocyte elastase this new substrate is 50 times more sensitive compared to succinyltrialanyl-p-nitroanilide. L-Pyroglutamyl-L-prolyl-L-valine-p-nitroanilide can also be used for the assay of granulocyte elastase inhibitors.

Chromogenic Compounds↗
PubMed2001

Substrate specificity and subsite mobility in T. aurantiacus xylanase 10A.

The substrate specificity of Thermoascus aurantiacus xylanase 10A (TAX) has been investigated both biochemically and structurally. High resolution crystallographic analyses at 291 K and 100 K of TAX complexes with xylobiose show that the ligand is in its alpha anomeric conformation and provide a rationale for specificity on p-nitrophenyl glycosides at the -1 and -2 subsites. Trp 275, which is disordered in uncomplexed structures, is stabilised by its interaction with xylobiose. Two structural subsets in family 10 are identified, which differ by the presence or absence of a short helical stretch in the eighth betaalpha-loop of the TIM barrel, the loop bearing Trp 275. This structural difference is discussed in the context of Trp 275 mobility and xylanase function.

Ascomycota↗
PubMedDate not supplied

[Substrate specificity of the biotransformation enzymes in a Nocardia erythropolis culture].

Substrate specificity of biotransformation enzymes of culture Nocardia erythropolis was studied. Products of transformation of cholesterol and three sterols of microbial origin: ergosterol, ergosta-5,7-dien-3 beta-ol and ergosta-7,22-dien-3 beta-ol was identified with a help of thin-layer chromatography, UV spectrophotometry and mass-spectrometry. It was established, that delta 22-bond in the side chains of sterols and delta 7-bond slows and delta 5-bond makes impossible cleavage of side chains of sterols.

Biotransformation↗
PubMed1987

The use of aminopeptidase substrate specificity profiles to identify leptospires.

Thirty reference Leptospira strains and twelve leptospire-like cultures were examined for aminopeptidase activity using twenty-two aminoacyl-beta-naphthylamide substrates. Aminopeptidase activity was demonstrated in extracts of each of the cultures and their substrate specificity profiles compared using several computer analysis procedures. The specificity profiles were consistent for each of the strains examined. Leptospira were readily differentiated from non Leptospira strains. This was supported by DNA base ratios calculated for the strains. Of the non Leptospira strains, seven appeared similar to the "L. illini" cultured included. Two of these strains, which included the strain "L. parva" sp. nov., formed a subgroup of this "L. illini" group. Substrate specificity profiles for the L. interrogans and L. biflexa strains examined were also different from each other.

Aminopeptidases↗
PubMed1994

Protease substrate specificity mapping using membrane-bound peptides.

A method is described for assessing the substrate specificity of proteases by screening for proteolytic activity against large numbers of peptides. All 400 possible peptides derived from the 20 common amino acids were synthesized on small membrane disks in the arrangement FTC-spacer-amino acid P1-amino acid P'1-spacer-membrane, where FTC is a chromophoric group. The disks are incubated simultaneously with the protease, resulting in cleavage of the peptide between the P1 and P'1 amino acids, and the absorbance of the released chromophore is measured as a function of time. As demonstrated for chymotrypsin and papain, plots of the resulting data present a perspective view of the amino acid preferences on both sides of the scissile bond. This technique is fast, requires relatively little enzyme, and can be extended to the systematic screening of longer peptides, including analogs with unnatural amino acids. It has potential use for characterizing the specificity of proteases, assessing the results of site-specific mutagenesis, and searching for optimal substrates and inhibitors.

Amino Acid Sequence↗
PubMed2002

Tetrachloroethene reductive dehalogenase of Dehalospirillum multivorans: substrate specificity of the native enzyme and its corrinoid cofactor.

The substrate specificity of the tetrachloroethene reductive dehalogenase of Dehalospirillum multivoransand its corrinoid cofactor were studied. Besides reduced methyl viologen, titanium(III) citrate could serve as electron donor for reductive dehalogenation of tetrachloroethene (PCE) and trichloroethene to cis-1,2-dichloroethene. In addition to chlorinated ethenes, chlorinated propenes were reductively dechlorinated solely by the native enzyme. trans-1,3-Dichloropropene, 1,1,3-trichloropropene and 2,3-dichloropropene were reduced to a mixture of mono-chloropropenes, 1,1-dichloropropene, and 2-chloropropene, respectively. Other halogenated compounds that were rapidly reduced by the enzyme were also dehalogenated abiotically by the heat-inactivated enzyme and by commercially available cyanocobalamin. The rate of this abiotic reaction was dependent on the number and type of halogen substituents and on the type of catalyst. The corrinoid cofactor purified from the tetrachloroethene dehalogenase of D. multivorans exhibited an activity about 50-fold higher than that of cyanocobalamin (vitamin B(12)) with trichloroacetate as electron acceptor, indicating that the corrinoid cofactor of the PCE dehalogenase is not cyanocobalamin. Corrinoids catalyzed the rapid dehalogenation of trichloroacetic acid. The rate was proportional to the amount of, e.g. cyanocobalamin; therefore, the reductive dehalogenation assay can be used for the sensitive and rapid quantification of this cofactor.

Catalysis↗
PubMed1991

Substrate specificity and activation mechanisms of collagenase from human rheumatoid synovium.

Substrate specificity studies of collagenase extracted from human rheumatoid synovium suggest that synovial pannus tissue overlying articular cartilage may not be particularly active in degradation of cartilage type II collagen, which, considering the poor inherent healing capacity of the articular hyaline cartilage, may exert a protective function against inadvertant tissue damage. Rheumatoid synovial tissue was also used to establish synovial fibroblast cell lines. Treatment of these cells in monolayer cultures with IL-1 leads to collagenase gene activation, increased collagenase production and an almost complete autoactivation of secreted collagenase. Interleukin-1 also activated stromelysin gene suggesting this as a possible mechanism effecting autoactivation. Latent human fibroblast and macrophage collagenase purified from culture medium were efficiently activated by phenylmercuric chloride but also by gold thioglucose, gold sodium thiomalate and HCIO. These new observations support the Cys73 switch activation mechanism. In contrast to neutrophil collagenase, the activation by gold(I) compounds and HCIO was associated with a change in the apparent molecular weight of the fibroblast procollagenase. In addition, gold(I) compounds rendered collagenase more susceptible to thermal denaturation. Thus the fibroblast-type interstitial collagenase, probably derived from fibroblast- and macrophage-like synoviocytes, seems to provide the predominant collagenolytic potential in human rheumatoid synovial tissue. Furthermore, the conditions in synovitis tissue may be such as to favor at least initial activation of collagenase synthesized and secreted in situ.

Arthritis, Rheumatoid↗
PubMed1996

Structure-function analysis of human alpha1,3-fucosyltransferase. Amino acids involved in acceptor substrate specificity.

A series of molecular biology experiments were carried out to identify the catalytic domain of two human alpha1,3/4-fucosyltransferases (fucosyltransferases (FucTs) III and V), and to identify amino acids that function in acceptor substrate binding. Sixty-one and 75 amino acids could be eliminated from the N terminus of FucTs III and V, respectively, without a significant loss of enzyme activity. In contrast, the truncation of one or more amino acids from the C terminus of FucT V resulted in a dramatic or total loss of enzyme activity. Results from the truncation experiments demonstrate that FucT III62-361 (containing amino acids 62-361) and FucT V76-374 (containing amino acids 76-374) are active, whereas shorter forms of the enzymes were inactive. The shortest, active forms of the enzymes are more than 93% identical at the predicted amino acid level, but have distinct acceptor substrate specificities. Thus, FucT III is an alpha1,4-fucosyltransferase, whereas FucT V is an alpha1,3-fucosyltransferase with disaccharide substrates. All but one of the amino acid sequence differences between the two proteins occur near their N terminus. Results obtained from domain swapping experiments demonstrated that the single amino acid sequence difference near the C terminus of these enzymes did not alter the enzyme's substrate specificity. However, swapping a region near the N terminus of the truncated form of FucT III into an homologous region in FucT V produced a protein with both alpha1,3- and alpha1,4-fucosyltransferase activity. This region contains 8 of the amino acid sequence differences that occur between the two proteins.

Amino Acid Sequence↗
PubMed2006

Substrate specificity of thermostable D-alanine-D-alanine ligase from Thermotoga maritima ATCC 43589.

D-Alanine-D-alanine ligase (Ddl) and its mutants maintain the biosynthesis of peptidoglycan, and the substrate specificity of Ddls partially affects the resistance mechanism of vancomycin-resistant enterococci. Through investigation of Ddls, Ddl from Thermotoga maritima ATCC 43589 showed novel characteristics, vis. thermostability up to 90 degrees C and broad substrate specificity toward 15 D-amino acids, particularly D-alanine, D-cysteine, and D-serine, in that order.

Enzyme Stability↗
PubMed2005

Transport mechanism and substrate specificity of human organic anion transporter 2 (hOat2 [SLC22A7]).

Human organic anion transporter 2 (hOat2[SLC22A7]) is highly expressed in the human liver. Although localization, gene expression, substrate specificity and transport mechanisms of other human Oat isoforms such as human Oat1 (hOat1), human Oat3 (hOat3) and human Oat4 (hOat4) have been elucidated, information concerning human Oat2 (hOat2) is less defined. The objective of this study was to provide further information on the transport mechanism and substrate specificity of hOat2. When expressed in Xenopus laevis oocytes, the transport of organic compounds mediated by hOat2 was not affected by the replacement of extracellular sodium with lithium, choline and mannitol. The uptake of estrone sulfate (ES) in hOat2-expressing oocytes was significantly trans-stimulated by preloading the oocytes with fumarate and succinate, but not glutarate. Moreover, we observed that hOat2 mediates the transport of bumetanide, ES, glutarate, dehydroepiandrosterone sulfate, allopurinol, prostaglandin E2, 5-fluorouracil, paclitaxel and L-ascorbic acid. These compounds are identified for the first time as hOat2 substrates. A wide range of structurally unrelated organic compounds inhibited the hOat2-mediated uptake of tetracycline, except for sulfobromophthalein. All of these findings indicate that hOat2 is a sodium-independent multi-specific organic anion/dimethyldicarboxylate exchanger. Our present findings thus provide further insights into the role of hOat2 in hepatic drug transport.

Animals↗