共查询到20条相似文献,搜索用时 15 毫秒
1.
Sharma N Oikonomopoulou K Ito K Renaux B Diamandis EP Hollenberg MD Rancourt DE 《Biological chemistry》2008,389(8):1097-1105
We constructed a random library of hexapeptides displayed on the surface of bacteriophage T7 to determine the substrate specificity of proteinases. The phage-displayed library was subjected to repeated rounds of biopanning with native implantation serine proteinase and recombinant human kallikrein-related peptidase 6 (KLK6) followed by selection and identification of putative substrates. For both enzymes, the results obtained demonstrate a preference for arginine and lysine at multiple positions in the recognition cleavage motif, confirming their previously reported trypsin-like substrate specificity. In the case of KLK6, there is also a pronounced presence of tryptophan within the cleaved peptide sequences, indicating its potential dual substrate specificity, acting as both a trypsin and chymotrypsin-like enzyme. 相似文献
2.
Paes Leme AF Escalante T Pereira JG Oliveira AK Sanchez EF Gutiérrez JM Serrano SM Fox JW 《Journal of Proteomics》2011,74(4):401-410
Both serine and metalloproteinases have been shown to play the role of toxins in the venoms of many snakes. Determination of the natural protein substrates of these toxins is an important feature in the toxinological characterization of these proteinases. Furthermore, characterization of their peptide bond specificity is of value for understanding active site preference of the proteinase associated with effective proteolysis as well as of use in the design of peptide substrates and inhibitor lead compounds. Typically the determination of peptide bond cleavage specificity of snake venom serine proteinases (SVSPs) and snake venom metalloproteinases (SVMPs) has been performed using limited sets of peptides or small oligopeptides as experimental substrates. Although this approach has yielded valuable data it is generally limited in scope due to the relatively small sets of substrates used to generate the consensus specificity sequences for these proteinases. In this study we use a large, plasma based, proteome-derived peptide library as substrates along with mass spectrometry to explore the peptide bond specificity of three PI SVMPs and one PIII SVMP to determine their individual peptide cleavage consensus sequences. All of the proteinases assayed displayed a clear preference for a leucine residue in the P1' site. Careful analysis of the specificity profiles of the SVMPs examined showed interesting differences in the preferences at the other P and P' sites suggesting functional differences between these proteinases. The PI SVMPs, leucurolysin-a, atrolysin C, and BaP1, showed preferences across the full P4 to P4' range whereas the PIII SVMP bothropasin showed a narrower range of preferences across the sites. In silico docking experiments with the experimentally derived consensus sequences as well as with comparison of the results to those in the literature regarding peptide bond specificity based on both peptide and protein substrates give rise to a fresh understanding of the specificity of these SVMPS and may serve as a foundation for future experiments to better elucidate their mechanism of action in the complex pathophysiology of snakebite envenomation. 相似文献
3.
Furlong ST Mauger RC Strimpler AM Liu YP Morris FX Edwards PD 《Bioorganic & medicinal chemistry》2002,10(11):3637-3647
Serine peptidases are a large, well-studied, and medically important class of peptidases. Despite the attention these enzymes have received, details concerning the substrate specificity of even some of the best known enzymes in this class are lacking. One approach to rapidly characterizing substrate specificity for peptidases is the use of positional scanning combinatorial substrate libraries. We recently synthesized such a library for enzymes with a preference for arginine at P1 and demonstrated the use of this library with thrombin (Edwards et al. Bioorg. Med. Chem. Lett. 2000, 10, 2291). In the present work, we extend these studies by demonstrating good agreement between the theroretical and measured content of portions of this library and by showing that the library permits rapid characterization of the substrate specificity of additional SA clan serine peptidases including factor Xa, tryptase, and trypsin. These results were consistent both with cleavage sites in natural substrates and cleavage of commercially available synthetic substrates. We also demonstrate that pH or salt concentration have a quantitative effect on the rate of cleavage of the pooled library substrates but that correct prediction of optimal substrates for the enzymes studied appeared to be independent of these parameters. These studies provide new substrate specificity data on an important class of peptidases and are the first to provide physical characterization of a peptidase substrate library. 相似文献
4.
A peptide library approach based on electrospray mass-spectrometric (ESI-MS) detection of phosphopeptides was designed for rapid and quantitative characterization of protein kinase specificity. The k(cat)/K(m) values for the protein kinase Cbeta (PKCbeta) were determined for a systematically varied set of individual substrate peptides in library mixtures by the ESI-MS method. The analysis revealed a complex structural specificity profile in positions around the phosphorylated serine with hydrophobic and/or basic residues being mostly preferred. On the basis of the kinetic parameters, a highly efficient peptide substrate for PKCbeta (K(m)value below 100 nM) FRRRRSFRRR and its alanine substituted pseudosubstrate-analog inhibitor (K(i) value of 76 nM) were designed. The quantitative specificity profiles obtained by the new approach contained more information about kinase specificity than the conventional substrate consensus motifs. The new method presents a promising basis for design of substrate-site directed peptide or peptidomimetic inhibitors of protein kinases. Second, highly specific substrates could be designed for novel applications such as high-throughput protein kinase activity screens on protein kinase chips. 相似文献
5.
Mouse granzyme B is a member of the chymotrypsin family of serine proteinases that has an unusual preference for cleavage of substrates following aspartate residues. We show here that granzyme B can be redesigned by a single amino acid substitution in one wall of the specificity pocket, arginine-226 to glutamate, to hydrolyze preferentially thioester substrates following basic amino acids. Amide substrates, however, were not hydrolyzed by the variant granzyme B. These results show that residue 226 is a primary determinant of granzyme B specificity and imply that additional structural components are required for catalysis of amide bonds. Molecular modeling indicated subtle variation in glutamate-226 orientation depending upon the state of protonation of the gamma-carboxylate, which may account for the secondary specificity of this enzyme for substrates containing phenylalanine. This represents the first example of electrostatic reversal of serine proteinase substrate specificity and suggests that residue 226 is a primary substrate specificity determinant in the granzyme B lineage of serine proteinases. 相似文献
6.
The number of known proteases is increasing at a tremendous rate as a consequence of genome sequencing projects. Although one can guess at the functions of these novel enzymes by considering sequence homology to known proteases, there is a need for new tools to rapidly provide functional information on large numbers of proteins. We describe a method for determining the cleavage site specificity of proteolytic enzymes that involves pooled sequencing of peptide library mixtures. The method was used to determine cleavage site motifs for six enzymes in the matrix metalloprotease (MMP) family. The results were validated by comparison with previous literature and by analyzing the cleavage of individually synthesized peptide substrates. The library data led us to identify the proteoglycan neurocan as a novel MMP-2 substrate. Our results indicate that a small set of libraries can be used to quickly profile an expanding protease family, providing information applicable to the design of inhibitors and to the identification of protein substrates. 相似文献
7.
Bell JK Goetz DH Mahrus S Harris JL Fletterick RJ Craik CS 《Nature structural biology》2003,10(7):527-534
The cell death-inducing serine protease granzyme A (GzmA) has a unique disulfide-linked quaternary structure. The structure of human GzmA bound to a tripeptide CMK inhibitor, determined at a resolution of 2.4 A, reveals that the oligomeric state contributes to substrate selection by limiting access to the active site for potential macromolecular substrates and inhibitors. Unlike other serine proteases, tetrapeptide substrate preferences do not correlate well with natural substrate cleavage sequences. This suggests that the context of the cleavage sequence within a macromolecular substrate imposes another level of selection not observed with the peptide substrates. Modeling of inhibitors bound to the GzmA active site shows that the dimer also contributes to substrate specificity in a unique manner by extending the active-site cleft. The crystal structure, along with substrate library profiling and mutagenesis, has allowed us to identify and rationally manipulate key components involved in GzmA substrate specificity. 相似文献
8.
9.
Specificity profiling of seven human tissue kallikreins reveals individual subsite preferences 总被引:2,自引:0,他引:2
Debela M Magdolen V Schechter N Valachova M Lottspeich F Craik CS Choe Y Bode W Goettig P 《The Journal of biological chemistry》2006,281(35):25678-25688
Human tissue kallikreins (hKs) form a family of 15 closely related (chymo)trypsin-like serine proteinases. These tissue kallikreins are expressed in a wide range of tissues including the central nervous system, the salivary gland, and endocrine-regulated tissues, such as prostate, breast, or testis, and may have diverse physiological functions. For several tissue kallikreins, a clear correlation has been established between expression and different types of cancer. For example, the prostate-specific antigen (PSA or hK3) serves as tumor marker and is used to monitor therapy response. Using a novel strategy, we have cloned, expressed in Escherichia coli or in insect cells, refolded, activated, and purified the seven human tissue kallikreins hK3/PSA, hK4, hK5, hK6, hK7, hK10, and hK11. Moreover, we have determined their extended substrate specificity for the nonprime side using a positional scanning combinatorial library of tetrapeptide substrates. hK3/PSA and hK7 exhibited a chymotrypsin-like specificity preferring large hydrophobic or polar residues at the P1 position. In contrast, hK4, hK5, and less stringent hK6 displayed a trypsin-like specificity with strong preference for P1-Arg, whereas hK10 and hK11 showed an ambivalent specificity, accepting both basic and large aliphatic P1 residues. The extended substrate specificity profiles are in good agreement with known substrate cleavage sites but also in accord with experimentally solved (hK4, hK6, and hK7) or modeled structures. The specificity profiles may lead to a better understanding of human tissue kallikrein functions and assist in identifying their physiological protein substrates as well as in designing more selective inhibitors. 相似文献
10.
Bown DP Wilkinson HS Jongsma MA Gatehouse JA 《Insect biochemistry and molecular biology》2004,34(4):305-320
Cysteine proteinases are the major class of enzymes responsible for digestive proteolysis in western corn rootworm (Diabrotica virgifera), a serious pest of maize. A larval gut extract hydrolysed typical cathepsin substrates, such as Z-phe-arg-AMC and Z-arg-arg-AMC, and hydrolysis was inhibited by Z-phe-tyr-DMK, specific for cathepsin L. A cDNA library representing larval gut tissue mRNA contained cysteine proteinase-encoding clones at high frequency. Sequence analysis of 11 cysteine proteinase cDNAs showed that 9 encoded cathepsin L-like enzymes, and 2 encoded cathepsin B-like enzymes. Three enzymes (two cathepsin L-like, DvRS5 and DvRS30, and one cathepsin B-like, DvRS40) were expressed as recombinant proteins in culture supernatants of the yeast Pichia pastoris. The cathepsin L-like enzymes were active proteinases, whereas the cathepsin B-like enzyme was inactive until treated with bovine trypsin. The amino acid residue in the S2 binding pocket, the major determinant of substrate specificity in cathepsin cysteine proteinases, predicted that the two cathepsin L-like enzymes, DvRS5 and DvRS30, should differ in substrate specificity, with the latter resembling cathepsin B in hydrolysing substrates with a positively charged residue at P2. This prediction was confirmed; DvRS5 only hydrolysed Z-phe-arg-AMC and not Z-arg-arg-AMC, whereas DvRS30 hydrolysed both substrates. The enzymes showed similar proteolytic activity towards peptide substrates. 相似文献
11.
O'Neill T Dwyer AJ Ziv Y Chan DW Lees-Miller SP Abraham RH Lai JH Hill D Shiloh Y Cantley LC Rathbun GA 《The Journal of biological chemistry》2000,275(30):22719-22727
The ataxia telangiectasia mutated (ATM) gene encodes a serine/threonine protein kinase that plays a critical role in genomic surveillance and development. Here, we use a peptide library approach to define the in vitro substrate specificity of ATM kinase activity. The peptide library analysis identified an optimal sequence with a central core motif of LSQE that is preferentially phosphorylated by ATM. The contributions of the amino acids surrounding serine in the LSQE motif were assessed by utilizing specific peptide libraries or individual peptide substrates. All amino acids comprising the LSQE sequence were critical for maximum peptide substrate suitability for ATM. The DNA-dependent protein kinase (DNA-PK), a Ser/Thr kinase related to ATM and important in DNA repair, was compared with ATM in terms of peptide substrate selectivity. DNA-PK was found to be unique in its preference of neighboring amino acids to the phosphorylated serine. Peptide library analyses defined a preferred amino acid motif for ATM that permits clear distinctions between ATM and DNA-PK kinase activity. Data base searches using the library-derived ATM sequence identified previously characterized substrates of ATM, as well as novel candidate substrate targets that may function downstream in ATM-directed signaling pathways. 相似文献
12.
Gurrinder Singh Bedi 《Preparative biochemistry & biotechnology》2013,43(2):85-104
Abstract The present investigation describes the comparative properties, particularly the substrate specificity of three kallikrein-like serine proteinases (I, II and III) purified from rat submandibular gland extract (Bedi, G.S., Prep. Biochem. 22, 67–81. 1992). The physico-chemical and immunological properties of three proteinases were compared by Western blot analysis, immunodiffusion, immuno-electrophoresis, amino terminal sequence analysis, molecular weight determination and isoelectric focusing. Detailed substrate specificity of these proteinases was determined using chromogenic substrates, synthetic peptides and native proteins. The chromogenic substrate tosyl-gly-pro-arg-pNA was hydrolyzed preferentially by Proteinase I. The replacement of pro at the P2 position with bulky hydrophobic residues phe and leu completely abolished the hydrolysis by Proteinase I. The hydrolysis of the chromogenic substrates by Proteinase II was also affected by the amino acid residue present at the P2 position in the order of pro>gly>val>leu>phe. Neither Proteinase I nor Proteinase II hydrolyzed substrates in which arg was replaced with lys at the P1 position. Proteinase III was reactive against all the chromogenic substrates with arg or lys at the P1 position. Synthetic polypeptides T-kinin-leu and insulin B chain were resistant to cleavage by both Proteinase I and II and were cleaved specifically at arg-X peptide bond by Proteinase III. Tonin-like activity of Proteinase II was confirmed by cleavage of the angiotensin 1–14 at phe-his linkage to generate two fragments DRVYIHPF and HLLVYS respectively. All three proteinases cleaved human high molecular weight kininogen but only Proteinase III could cleave T-kininogen. Proteinase III was also reactive towards human and bovine fibronectin, fibrinogen and gelatin. Several other salivary and serum proteins were resistant to cleavage by these proteinases. Although the three enzymes are immunologically related, they differ with respect to size, isoelectric point, amino terminal sequence and inhibition profile. 相似文献
13.
Hanzon J Smirnoff P Applebaum SW Mattoo AK Birk Y 《Archives of biochemistry and biophysics》2003,410(1):83-88
Two unique serine proteinase isoenzymes (LmHP-1 and LmHP-2) were isolated from the hemolymph of African migratory locust (Locusta migratoria migratorioides) nymphs. Both have a molecular mass of about 23 kDa and are activated by thiol-reducing agents. PMSF abolishes enzymes activity only after thiol activation, while the cysteine proteinase inhibitors E-64, iodoacetamide, and heavy metals fail to inhibit the thiol-activated enzymes. The N-terminal sequence was determined for the more-abundant LmHP-2 isoenzyme. It exhibits partial homology to that of other insect serine proteinases and similar substrate specificity and inhibition by the synthetic and protein trypsin inhibitors pABA, TLCK, BBI, and STI. The locust trypsins LmHP-1 and LmHP-2 constitute a new category of serine proteases wherein the active site of the enzyme is exposed by thiol activation without cleavage of peptide bonds. 相似文献
14.
Evolutionary optimization of peptide substrates for proteases that exhibit rapid hydrolysis kinetics
Kevin T. Boulware Abeer Jabaiah Patrick S. Daugherty 《Biotechnology and bioengineering》2010,106(3):339-346
Protease cleavage site recognition motifs can be identified using protease substrate discovery methodologies, but typically exhibit non‐optimal specificity and activity. To enable evolutionary optimization of substrate cleavage kinetics, a two‐color cellular library of peptide substrates (CLiPS) methodology was developed. Two‐color CLiPS was applied to identify peptide substrates for the tobacco etch virus (TEV) protease from a random pentapeptide library, which were then optimized by screening of a focused, extended substrate library. Quantitative library screening yielded seven amino acid substrates exhibiting rapid hydrolysis by TEV protease and high sequence similarity to the native seven‐amino‐acid substrate, with a strong consensus of EXLYΦQG. Comparison of hydrolysis rates for a family of closely related substrates indicates that the native seven‐residue TEV substrate co‐evolved with TEV protease to facilitate highly efficient hydrolysis. Consensus motifs revealed by screening enabled database identification of a family of related, putative viral protease substrates. More generally, our results suggest that substrate evolution using CLiPS may be useful for optimizing substrate selectivity and activity to enable the design of more effective protease activity probes, molecular imaging agents, and prodrugs. Biotechnol. Bioeng. 2010; 106: 339–346. © 2010 Wiley Periodicals, Inc. 相似文献
15.
C D Robertson M J North B C Lockwood G H Coombs 《Journal of general microbiology》1990,136(5):921-925
A method comprising enzyme separation by SDS-PAGE and subsequent use of peptidyl aminomethylcoumarins as substrates has been used to study proteinases of the protozoan parasite Trypanosoma brucei. The application of this method has allowed investigation of the substrate specificities of individual proteinases in cell lysates without the need for enzyme purification. The results show that T. brucei contains a group of cysteine proteinases, probably four in number, with substrate and inhibitor specificities similar to those of cathepsin L. A second group of proteinases, larger enzymes with significantly different substrate specificities and sensitivity to inhibitors, was also detected. Peptidyl diazomethanes inhibited the cysteine proteinases and also parasite growth, offering promise that peculiarities in the substrate specificity of trypanosomal cysteine proteinases could be exploited by compounds of this type. 相似文献
16.
Choe Y Leonetti F Greenbaum DC Lecaille F Bogyo M Brömme D Ellman JA Craik CS 《The Journal of biological chemistry》2006,281(18):12824-12832
The substrate specificities of papain-like cysteine proteases (clan CA, family C1) papain, bromelain, and human cathepsins L, V, K, S, F, B, and five proteases of parasitic origin were studied using a completely diversified positional scanning synthetic combinatorial library. A bifunctional coumarin fluorophore was used that facilitated synthesis of the library and individual peptide substrates. The library has a total of 160,000 tetrapeptide substrate sequences completely randomizing each of the P1, P2, P3, and P4 positions with 20 amino acids. A microtiter plate assay format permitted a rapid determination of the specificity profile of each enzyme. Individual peptide substrates were then synthesized and tested for a quantitative determination of the specificity of the human cathepsins. Despite the conserved three-dimensional structure and similar substrate specificity of the enzymes studied, distinct amino acid preferences that differentiate each enzyme were identified. The specificities of cathepsins K and S partially match the cleavage site sequences in their physiological substrates. Capitalizing on its unique preference for proline and glycine at the P2 and P3 positions, respectively, selective substrates and a substrate-based inhibitor were developed for cathepsin K. A cluster analysis of the proteases based on the complete specificity profile provided a functional characterization distinct from standard sequence analysis. This approach provides useful information for developing selective chemical probes to study protease-related pathologies and physiologies. 相似文献
17.
Engineering of the Lactococcus lactis serine proteinase by construction of hybrid enzymes 总被引:7,自引:0,他引:7
P Vos I J Boerrigter G Buist A J Haandrikman M Nijhuis M B de Reuver R J Siezen G Venema W M de Vos J Kok 《Protein engineering》1991,4(4):479-484
Plasmids containing wild-type and hybrid proteinase genes were constructed from DNA fragments of the prtP genes of Lactococcus lactis strains Wg2 and SK11. These plasmids were introduced into the plasmid-free strain L. lactis MG1363. The serine proteinases produced by these L. lactis strains were isolated, and their cleavage specificity and rate towards alpha s1- and beta-casein was investigated. The catalytic properties of both the SK11 and Wg2 proteinases, which differ in 44 out of 1902 amino acid residues, could be changed dramatically by the reciprocal exchange of specific fragments between the two enzymes. As a result, various L. lactis strains were constructed having new proteolytic properties that differ from those of the parental strains. Furthermore, two segments in the proteinase could be identified that contribute significantly to the cleavage specificity towards casein; within these two segments, several amino acid residues were identified that are important for substrate cleavage rate and specificity. The results also indicate that the lactococcal proteinase has an additional domain involved in substrate binding compared with the related subtilisins. This suggests that the 200 kd L. lactis proteinase may be the representative of a new subclass of subtilisin-like enzymes. 相似文献
18.
Angela M. Smith Andrew J. Dowd Mary Heffernan Colin D. Robertson John P. Dalton 《International journal for parasitology》1993,23(8):977-983
Adult Fasciola hepatica secrete a cysteine proteinase capable of cleaving host IgG close to the papain cleaving site. The proteinase was separated by size permeation chromatography. Gelatinsubstrate polyacrylamide gel electrophoresis analysis revealed that the proteinase migrates as 6 proteolytic bands in the apparent molecular size range 60–90 kDa. Based on pH profiles of activity, inhibition studies using diethylpyrocarbonate and the diazomethylketone Z-phe-ala-CHN2, and characterising the substrate specificity of the enzymes using fluorogenic peptide substrates we have shown that the 60–90-kDa proteinases are cathepsin L-Iike proteinases. 相似文献
19.
Effect of N-methylation on the modulation by synthetic peptides of the activity of the complement-factor-B-derived serine proteinase CVFBb.
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Although they share the active-site catalytic triad of less-specific enzymes such as trypsin and chymotrypsin, the serine proteinases of the complement and coagulation cascades each cleave a highly restricted set of substrates. Peptides with sequences similar to that at which C3 is cleaved by the alternative-pathway complement proteinase CVFBb were synthesized by solid-phase methodology and examined for their effects on the activity of this enzyme as measured by three different types of assays. It was found that a peptide methylated at the scissile bond was a far more effective inhibitor of the cleavage of the protein substrate C5 and of the lysis of guinea-pig erythrocytes by the alternative pathway than was the equivalent unmethylated peptide. Whereas the unmethylated peptide inhibited cleavage of the peptide substrate, the methylated peptide actually stimulated cleavage in this assay. This stimulation was found to be due to a 2.8-fold increase in kcat; the dissociation constant for the substrate was not altered significantly. One model consistent with this behaviour is that the binding of the activator peptide in the extended substrate-recognition region stabilizes a catalytically more active conformation of the active site. A small peptide substrate may have access to such an activated active site, whereas the larger substrate, C5, may be excluded from the site. These results demonstrate that the observed effect of a given compound on activity of an enzyme with an extended substrate-recognition region may depend upon the substrate. 相似文献
20.
E. V. Lavrenteva Ya. E. Dunaevsky L. P. Kozyreva A. A. Radnagurueva B. B. Namsaraev 《Applied Biochemistry and Microbiology》2010,46(6):580-585
Influence of nitrogen source on proteinases synthesis in aerobic alkalotolerant and halotolerant bacteria from soda-salt lakes
of Transbaikalia was studied. Maximal accumulation of proteinases was revealed on medium with peptones. Introduction of various
sources of nitrogen in the medium did not result in increase of enzyme activity in cultural liquid. It was indicated that
secreting proteinases of the studied bacteria strains possess narrow substrate specificity, hydrolyze proteins and n-nitroanilide substrates have maximal activity during GlpAALpNA hydrolysis. Data of inhibitory analysis and substrate specificity
of studied extracellular enzymes indicate that they belong to a class of serine proteinases of subtilisin-like type. 相似文献