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1.
2.
Anne Davy Ib Svendsen Susanne O. S?rensen Mikael Blom S?rensen Jacques Rouster Morten Meldal David J. Simpson Verena Cameron-Mills 《Plant physiology》1998,117(1):255-261
The cysteine endoproteases (EP)-A and EP-B were purified from green barley (Hordeum vulgare L.) malt, and their identity was confirmed by N-terminal amino acid sequencing. EP-B cleavage sites in recombinant type-C hordein were determined by N-terminal amino acid sequencing of the cleavage products, and were used to design internally quenched, fluorogenic peptide substrates. Tetrapeptide substrates of the general formula 2-aminobenzoyl-P2-P1-P1′-P2′-tyrosine(NO2)-aspartic acid, in which cleavage occurs between P1 and P1′, showed that the cysteine EPs preferred phenylalanine, leucine, or valine at P2. Arginine was preferred to glutamine at P1, whereas proline at P2, P1, or P1′ greatly reduced substrate kinetic specificity. Enzyme cleavage of C hordein was mainly determined by the primary sequence at the cleavage site, because elongation of substrates, based on the C hordein sequence, did not make them more suitable substrates. Site-directed mutagenesis of C hordein, in which serine or proline replaced leucine, destroyed primary cleavage sites. EP-A and EP-B were both more active than papain, mostly because of their much lower Km values. 相似文献
3.
Chunhui Liu Juzheng Sheng Juno M. Krahn Lalith Perera Yongmei Xu Po-Hung Hsieh Wenfang Dou Jian Liu Lars C. Pedersen 《The Journal of biological chemistry》2014,289(19):13407-13418
Heparan sulfate (HS) is an abundant polysaccharide in the animal kingdom with essential
physiological functions. HS is composed of sulfated saccharides that are biosynthesized through a
complex pathway involving multiple enzymes. In vivo regulation of this process
remains unclear. HS 2-O-sulfotransferase (2OST) is a key enzyme in this pathway.
Here, we report the crystal structure of the ternary complex of 2OST, 3′-phosphoadenosine
5′-phosphate, and a heptasaccharide substrate. Utilizing site-directed mutagenesis and
specific oligosaccharide substrate sequences, we probed the molecular basis of specificity and 2OST
position in the ordered HS biosynthesis pathway. These studies revealed that Arg-80, Lys-350, and
Arg-190 of 2OST interact with the N-sulfo groups near the modification site,
consistent with the dependence of 2OST on N-sulfation. In contrast,
6-O-sulfo groups on HS are likely excluded by steric and electrostatic repulsion
within the active site supporting the hypothesis that 2-O-sulfation occurs prior to
6-O-sulfation. Our results provide the structural evidence for understanding the
sequence of enzymatic events in this pathway. 相似文献
4.
5.
Binding of substrates into the active site, often through complementarity of shapes and charges, is central to the specificity of an enzyme. In many cases, substrate binding induces conformational changes in the active site, promoting specific interactions between them. In contrast, non-substrates either fail to bind or do not induce the requisite conformational changes upon binding and thus no catalysis occurs. In principle, both lock and key and induced-fit binding can provide specific interactions between the substrate and the enzyme. In this study, we present an interesting case where cofactor binding pre-tunes the active site geometry to recognize only the cognate substrates. We illustrate this principle by studying the substrate binding and kinetic properties of Xylose Reductase from Debaryomyces hansenii (DhXR), an AKR family enzyme which catalyzes the reduction of carbonyl substrates using NADPH as co-factor. DhXR reduces D-xylose with increased specificity and shows no activity towards “non-substrate” sugars like L-rhamnose. Interestingly, apo-DhXR binds to D-xylose and L-rhamnose with similar affinity (Kd∼5.0–10.0 mM). Crystal structure of apo-DhXR-rhamnose complex shows that L-rhamnose is bound to the active site cavity. L-rhamnose does not bind to holo-DhXR complex and thus, it cannot competitively inhibit D-xylose binding and catalysis even at 4–5 fold molar excess. Comparison of Kd values with Km values reveals that increased specificity for D-xylose is achieved at the cost of moderately reduced affinity. The present work reveals a latent regulatory role for cofactor binding which was previously unknown and suggests that cofactor induced conformational changes may increase the complimentarity between D-xylose and active site similar to specificity achieved through induced-fit mechanism. 相似文献
6.
A Determinant of Substrate Specificity Predicted from the
Acyl-Acyl Carrier Protein Desaturase of Developing
Cat''s Claw
Seed 总被引:1,自引:0,他引:1
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Cat's claw (Doxantha unguis-cati L.) vine accumulates nearly 80% palmitoleic acid (16:1Δ9) plus cis-vaccenic acid (18:1Δ11) in its seed oil. To characterize the biosynthetic origin of these unusual fatty acids, cDNAs for acyl-acyl carrier protein (acyl-ACP) desaturases were isolated from developing cat's claw seeds. The predominant acyl-ACP desaturase cDNA identified encoded a polypeptide that is closely related to the stearoyl (Δ9–18:0)-ACP desaturase from castor (Ricinis communis L.) and other species. Upon expression in Escherichia coli, the cat's claw polypeptide functioned as a Δ9 acyl-ACP desaturase but displayed a distinct substrate specificity for palmitate (16:0)-ACP rather than stearate (18:0)-ACP. Comparison of the predicted amino acid sequence of the cat's claw enzyme with that of the castor Δ9–18:0-ACP desaturase suggested that a single amino acid substitution (L118W) might account in large part for the differences in substrate specificity between the two desaturases. Consistent with this prediction, conversion of leucine-118 to tryptophan in the mature castor Δ9–18:0-ACP desaturase resulted in an 80-fold increase in the relative specificity of this enzyme for 16:0-ACP. The alteration in substrate specificity observed in the L118W mutant is in agreement with a crystallographic model of the proposed substrate-binding pocket of the castor Δ9–18:0-ACP desaturase. 相似文献
7.
Rinrada Luechapanichkul Xianwen Chen Hashem A. Taha Shubham Vyas Xiaoyan Guan Michael A. Freitas Christopher M. Hadad Dehua Pei 《The Journal of biological chemistry》2013,288(9):6498-6510
Vaccinia VH1-related (VHR) is a dual specificity phosphatase that consists of only a single catalytic domain. Although several protein substrates have been identified for VHR, the elements that control the in vivo substrate specificity of this enzyme remain unclear. In this work, the in vitro substrate specificity of VHR was systematically profiled by screening combinatorial peptide libraries. VHR exhibits more stringent substrate specificity than classical protein-tyrosine phosphatases and recognizes two distinct classes of Tyr(P) peptides. The class I substrates are similar to the Tyr(P) motifs derived from the VHR protein substrates, having sequences of (D/E/φ)(D/S/N/T/E)(P/I/M/S/A/V)pY(G/A/S/Q) or (D/E/φ)(T/S)(D/E)pY(G/A/S/Q) (where φ is a hydrophobic amino acid and pY is phosphotyrosine). The class II substrates have the consensus sequence of (V/A)P(I/L/M/V/F)X1–6pY (where X is any amino acid) with V/A preferably at the N terminus of the peptide. Site-directed mutagenesis and molecular modeling studies suggest that the class II peptides bind to VHR in an opposite orientation relative to the canonical binding mode of the class I substrates. In this alternative binding mode, the Tyr(P) side chain binds to the active site pocket, but the N terminus of the peptide interacts with the carboxylate side chain of Asp164, which normally interacts with the Tyr(P) + 3 residue of a class I substrate. Proteins containing the class II motifs are efficient VHR substrates in vitro, suggesting that VHR may act on a novel class of yet unidentified Tyr(P) proteins in vivo. 相似文献
8.
Catherine E. Jessop Timothy J. Tavender Rachel H. Watkins Joseph E. Chambers Neil J. Bulleid 《The Journal of biological chemistry》2009,284(4):2194-2202
The formation of disulfides within proteins entering the secretory pathway
is catalyzed by the protein disulfide isomerase family of endoplasmic
reticulum localized oxidoreductases. One such enzyme, ERp57, is thought to
catalyze the isomerization of non-native disulfide bonds formed in
glycoproteins with unstructured disulfide-rich domains. Here we investigated
the mechanism underlying ERp57 specificity toward glycoprotein substrates and
the interdependence of ERp57 and the calnexin cycle for their correct folding.
Our results clearly show that ERp57 must be physically associated with the
calnexin cycle to catalyze isomerization reactions with most of its
substrates. In addition, some glycoproteins only require ERp57 for correct
disulfide formation if they enter the calnexin cycle. Hence, the specificity
of ER oxidoreductases is not only determined by the physical association of
enzyme and substrate but also by accessory factors, such as calnexin and
calreticulin in the case of ERp57. These conclusions suggest that the calnexin
cycle has evolved with a specialized oxidoreductase to facilitate native
disulfide formation in complex glycoproteins.The ability to form disulfide bonds within proteins entering the secretory
pathway is essential for cell survival and occurs within the endoplasmic
reticulum (ER).3 For
proteins with few disulfides, the process can be catalyzed by oxidation of
cysteine residues to form the correct, native disulfide; however, for proteins
with several disulfides, an isomerization reaction is also required to correct
non-native disulfides formed following oxidation
(1). Both these reactions are
catalyzed by a group of ER-resident proteins that belong to the protein
disulfide isomerase (PDI) family, which comprises over 17 members
(2). It is well established
that PDI and several other family members are able to catalyze the formation
and isomerization of disulfides in vitro, although the exact function
of each of the family members in vivo is unknown. It is still an open
question as to whether they all catalyze similar reactions and have distinct
substrate specificities or whether they have distinct enzymatic functions
related to the breaking and formation of disulfides.For one member of the PDI family, the function and substrate specificity is
a little clearer. ERp57 has been shown previously to interact specifically
with glycoproteins during their folding
(3). The enzyme is physically
associated with either calnexin or calreticulin
(4) and is therefore ideally
placed to catalyze correct disulfide formation within proteins entering the
calnexin/calreticulin cycle (referred to subsequently just as the calnexin
cycle). In addition, the ability of ERp57 to catalyze the refolding of
substrates in vitro is greatly enhanced if the substrate is bound to
calnexin (5). Recently,
substrates for the reduction or isomerization reaction catalyzed by ERp57 have
been identified by trapping mixed disulfides between enzyme and substrate
(6). Strikingly, there was an
overrepresentation of substrate proteins with cysteine-rich domains containing
little secondary structure, suggesting that the main function of ERp57 is in
the isomerization of non-native disulfides. ERp57 has also been shown to
function independently from the calnexin cycle. It is a component of the MHC
class I loading complex where it forms a disulfide-linked complex with tapasin
and is thought to either stabilize the complex or facilitate correct assembly
of class I molecules (7,
8). Recently, ERp57 has been
demonstrated to isomerize interchain disulfides in the major capsid protein,
VP1, of simian virus 40 (9).
The ability to dissociate VP1 pentamers by ERp57 does not require the
substrate to interact with the calnexin cycle. Hence, it is still unclear how
ERp57 recognizes its substrates, and in particular, whether this recognition
is solely determined by an interaction with the calnexin cycle.The recognition of substrates by PDI is somewhat clearer in that one
particular domain within the protein (the b′ domain) has been shown to
be primarily responsible for substrate recognition and peptide binding
(10). The corresponding domain
within ERp57 has been shown to be responsible for interaction with the
calnexin cycle (11),
suggesting that for ERp57, substrate recognition must occur outside this
domain or is determined solely by substrate interaction with calnexin via its
oligosaccharide side chain. Hence, the aim of our study was to evaluate the
necessity of the calnexin cycle both for ERp57 to recognize its substrates and
for correct folding of glycoproteins. ERp57 was found to be required for the
efficient folding of one substrate, influenza virus hemagglutinin (HA), but
only when it entered the calnexin cycle. HA did not require ERp57 to fold if
it was blocked from entering the calnexin cycle. In contrast, β1-integrin
does not fold efficiently either if ERp57 was depleted or if ERp57 is blocked
from entering the calnexin cycle
(6). Although ERp57 may be
dispensable for the folding of some glycoproteins, the interaction with
calnexin commits them to an ERp57-dependent fate. We also found that the
majority of ERp57 substrates need to enter the calnexin cycle to be acted upon
by the enzyme, demonstrating that substrate specificity is primarily dependent
upon substrate entry into the calnexin cycle. 相似文献
9.
The presenilin/γ-secretase complex, an unusual intramembrane aspartyl protease, plays an essential role in cellular signaling and membrane protein turnover. Its ability to liberate numerous intracellular signaling proteins from the membrane and also mediate the secretion of amyloid-β protein (Aβ) has made modulation of γ-secretase activity a therapeutic goal for cancer and Alzheimer disease. Although the proteolysis of the prototypical substrates Notch and β-amyloid precursor protein (APP) has been intensely studied, the full spectrum of substrates and the determinants that make a transmembrane protein a substrate remain unclear. Using an unbiased approach to substrate identification, we surveyed the proteome of a human cell line for targets of γ-secretase and found a relatively small population of new substrates, all of which are type I transmembrane proteins but have diverse biological roles. By comparing these substrates to type I proteins not regulated by γ-secretase, we determined that besides a short ectodomain, γ-secretase requires permissive transmembrane and cytoplasmic domains to bind and cleave its substrates. In addition, we provide evidence for at least two mechanisms that can target a substrate for γ cleavage: one in which a substrate with a short ectodomain is directly cleaved independent of sheddase association, and a second where a substrate requires ectodomain shedding to instruct subsequent γ-secretase processing. These findings expand our understanding of the mechanisms of substrate selection as well as the diverse cellular processes to which γ-secretase contributes. 相似文献
10.
Andreas Naegeli Ga?lle Michaud Mario Schubert Chia-Wei Lin Christian Lizak Tamis Darbre Jean-Louis Reymond Markus Aebi 《The Journal of biological chemistry》2014,289(35):24521-24532
N-Linked protein glycosylation is a very common post-translational modification that can be found in all kingdoms of life. The classical, highly conserved pathway entails the assembly of a lipid-linked oligosaccharide and its transfer to an asparagine residue in the sequon NX(S/T) of a secreted protein by the integral membrane protein oligosaccharyltransferase. A few species in the class of γ-proteobacteria encode a cytoplasmic N-glycosylation system mediated by a soluble N-glycosyltransferase (NGT). This enzyme uses nucleotide-activated sugars to modify asparagine residues with single monosaccharides. As these enzymes are not related to oligosaccharyltransferase, NGTs constitute a novel class of N-glycosylation catalyzing enzymes. To characterize the NGT-catalyzed reaction, we developed a sensitive and quantitative in vitro assay based on HPLC separation and quantification of fluorescently labeled substrate peptides. With this assay we were able to directly quantify glycopeptide formation by Actinobacillus pleuropneumoniae NGT and determine its substrate specificities: NGT turns over a number of different sugar donor substrates and allows for activation by both UDP and GDP. Quantitative analysis of peptide substrate turnover demonstrated a strikingly similar specificity as the classical, oligosaccharyltransferase-catalyzed N-glycosylation, with NX(S/T) sequons being the optimal NGT substrates. 相似文献
11.
Developmental Expression and Substrate Specificities of Alfalfa
Caffeic Acid 3-O-Methyltransferase and
Caffeoyl
Coenzyme A 3-O-Methyltransferase in
Relation to
Lignification 总被引:2,自引:0,他引:2
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Kentaro Inoue Vincent J.H. Sewalt G. Murray Ballance Weiting Ni Cornelia Stürzer Richard A. Dixon 《Plant physiology》1998,117(3):761-770
12.
Masao Fujimoto Akira Kuninaka Hiroshi Yoshino 《Bioscience, biotechnology, and biochemistry》2013,77(9):1555-1561
Nuclease P1 cleaved substantially all phosphodiester bonds in rRNA, tRNA, poly(I), poly(U), poly(A), poly(C), poly(G), poly(I)·poly(C), native DNA and heat-denatured DNA to produce exclusively 5′-mononucleotides. Single-stranded polynucleotides were much more susceptible than double-stranded ones. Influence of pH and ionic strength on the hydrolysis rate significantly varied with the kind of polynucleotides. The enzyme also hydrolyzed 3′-phosphomonoester bonds in 3′-AMP, 3′-GMP, 3′-UMP, 3′-CMP, 3′-dAMP, 3′-dGMP, 3′-dCMP and 3′-dTMP. Ribonucleoside 3′-monophosphates were hydrolyzed 20 to 50 times faster than the corresponding 3′-deoxyribonucleotides. Base preference of the enzyme for 3′-ribonucleotides was in the order of G>A>C≧U, whereas that for 3′-deoxyribo-nucleotides was in the order of C≧T>A≧G. The 3′-phosphomonoester bonds in nucleoside 3′, 5′-diphosphates, coenzyme A and dinucleotides bearing 3′-phosphate were hydrolyzed at a rate similar to that for the corresponding 3′-mononucleotides. Adenosine 2′-monophosphate was highly resistant, being split at less than 1/3,000 the rate at which 3′-AMP was split. 相似文献
13.
The enzymes belonging to the cutinase family are serine enzymes active on a large panel of substrates such as cutin, triacylglycerols, and phospholipids. In the M. tuberculosis H37Rv genome, seven genes coding for cutinase-like proteins have been identified with strong immunogenic properties suggesting a potential role as vaccine candidates. Two of these enzymes which are secreted and highly homologous, possess distinct substrates specificities. Cfp21 is a lipase and Cut4 is a phospholipase A2, which has cytotoxic effects on macrophages. Structural overlay of their three-dimensional models allowed us to identify three areas involved in the substrate binding process and to shed light on this substrate specificity. By site-directed mutagenesis, residues present in these Cfp21 areas were replaced by residues occurring in Cut4 at the same location. Three mutants acquired phospholipase A1 and A2 activities and the lipase activities of two mutants were 3 and 15 fold greater than the Cfp21 wild type enzyme. In addition, contrary to mutants with enhanced lipase activity, mutants that acquired phospholipase B activities induced macrophage lysis as efficiently as Cut4 which emphasizes the relationship between apparent phospholipase A2 activity and cytotoxicity. Modification of areas involved in substrate specificity, generate recombinant enzymes with higher activity, which may be more immunogenic than the wild type enzymes and could therefore constitute promising candidates for antituberculous vaccine production. 相似文献
14.
A constitutive peptide lactonase from Actinoplanes missouriensis hydrolyzed echinomycin, stendomycin, thiostrepton, vernamycin B, staphylomycin S, and etamycin. An induced lactonase hydrolyzed actinomycin but not the other peptide lactones. 相似文献
15.
16.
《Bioscience, biotechnology, and biochemistry》2013,77(10):2104-2108
The substrate specificity of cucumisin [EC 3.4.21.25] was identified by the use of the synthetic peptide substrates Leum-Pro-Glu-Ala-Leun (m=0-4, n=0-3). Neither Pro-Glu-Ala-Leu (m=0) nor Leu-Pro-Glu-Ala (n=0) was cleaved by cucumisin, however other analogus peptides were cleaved between Glu-Ala. The hydrolysis rates of Leum-Pro-Glu-Ala-Leu increased with the increase of m=1 to 2 and 3, but was however, essentially same with the increase of m=3 to 4. Similarly, the hydrolysis rates of Leu-Leu-Pro-Glu-Ala-Leun increased with the increase of n=0 to 1 and 2, but was essentially same with the increase of n=2 to 3. Then, it was concluded that cucumisin has a S5-S3′ subsite length. In order to identify the substrate specificity at P1 position, Leu-Leu-Pro-X-Ala-Leu (X; Gly, Ala, Val, Leu, Ile, Pro, Asp, Glu, Lys, Arg, Asn, Gln, Phe, Tyr, Ser, Thr, Met, Trp, His) were synthesized and digested by cucumisin. Cucumisin showed broad specificity at the P1 position. However, cucumisin did not cleave the C-terminal side of Gly, Ile, Pro, and preferred Leu, Asn, Gln, Thr, and Met, especially Met. Moreover, the substrates, Leu-Leu-Pro-Glu-Y-Leu (Y; Gly, Ala, Ser, Leu, Val, Glu, Lys, Phe) were synthesized and digested by cucumisin. Cucumisin did not cleave the N-terminal side of Val but preferred Gly, Ser, Ala, and Lys especially Ser. The specificity of cucumisin for naturally occurring peptides does not agree strictly with the specificity obtained by synthetic peptides at the P1 or P1′ position alone, but it becomes clear that the most of the cleavage sites on naturally occurring peptides by cucumisin contain suitable amino acid residues at P1 and (or) P1′ positions. Moreover, cucumisin prefers Pro than Leu at P2 position, indicating that the specificity at P2 position differs from that of papain. 相似文献
17.
Dieter Demon Petra Van Damme Tom Vanden Berghe Annelies Deceuninck Joost Van Durme Jelle Verspurten Kenny Helsens Francis Impens Magdalena Wejda Joost Schymkowitz Frederic Rousseau Annemieke Madder Jo?l Vandekerckhove Wim Declercq Kris Gevaert Peter Vandenabeele 《Molecular & cellular proteomics : MCP》2009,8(12):2700-2714
Caspase-3 and -7 are considered functionally redundant proteases with similar proteolytic specificities. We performed a proteome-wide screen on a mouse macrophage lysate using the N-terminal combined fractional diagonal chromatography technology and identified 46 shared, three caspase-3-specific, and six caspase-7-specific cleavage sites. Further analysis of these cleavage sites and substitution mutation experiments revealed that for certain cleavage sites a lysine at the P5 position contributes to the discrimination between caspase-7 and -3 specificity. One of the caspase-7-specific substrates, the 40 S ribosomal protein S18, was studied in detail. The RPS18-derived P6–P5′ undecapeptide retained complete specificity for caspase-7. The corresponding P6–P1 hexapeptide still displayed caspase-7 preference but lost strict specificity, suggesting that P′ residues are additionally required for caspase-7-specific cleavage. Analysis of truncated peptide mutants revealed that in the case of RPS18 the P4–P1 residues constitute the core cleavage site but that P6, P5, P2′, and P3′ residues critically contribute to caspase-7 specificity. Interestingly, specific cleavage by caspase-7 relies on excluding recognition by caspase-3 and not on increasing binding for caspase-7.Caspases, a family of evolutionarily conserved proteases, mediate apoptosis, inflammation, proliferation, and differentiation by cleaving many cellular substrates (1–3). The apoptotic initiator caspases (caspase-8, -9, and -10) are activated in large signaling platforms and propagate the death signal by cleavage-induced activation of executioner caspase-3 and -7 (4, 5). Most of the cleavage events occurring during apoptosis have been attributed to the proteolytic activity of these two executioner caspases, which can act on several hundreds of proteins (2, 3, 6, 7). The substrate degradomes of the two main executioner caspases have not been determined but their identification is important to gaining greater insight in their cleavage specificity and biological functions.The specificity of caspases was rigorously profiled by using combinatorial tetrapeptide libraries (8), proteome-derived peptide libraries (9), and sets of individual peptide substrates (10, 11). The results of these studies indicate that specificity motifs for caspase-3 and -7 are nearly indistinguishable with the canonical peptide substrate, DEVD, used to monitor the enzymatic activity of both caspase-3 and -7 in biological samples. This overlap in cleavage specificity is manifested in their generation of similar cleavage fragments from a variety of apoptosis-related substrates such as inhibitor of caspase-activated DNase, keratin 18, PARP,1 protein-disulfide isomerase, and Rho kinase I (for reviews, see Refs. 2, 3, and 7). This propagated the view that these two caspases have completely redundant functions during apoptosis. Surprisingly, mice deficient in one of these caspases (as well as mice deficient in both) have distinct phenotypes. Depending on the genetic background of the mice, caspase-3-deficient mice either die before birth (129/SvJ) or develop almost normally (C57BL/6J) (12–14). This suggests that dynamics in the genetic background, such as increased caspase-7 expression, compensate for the functional loss of caspase-3 (15). In the C57BL/6J background, caspase-7 single deficient mice are also viable, whereas caspase-3 and -7 double deficient mice die as embryos, further suggesting redundancy (12–14). However, because caspase-3 and -7 probably arose from gene duplication between the Cephalochordata-Vertebrata diversion (16), they might have acquired different substrate specificities during evolution. Caspase-3 and -7 do exhibit different activities on a few arbitrarily identified natural substrates, including BID, X-linked inhibitor of apoptosis protein, gelsolin, caspase-6, ataxin-7, and co-chaperone p23 (17–20). In addition, caspase-3 generally cleaves more substrates during apoptosis than caspase-7 and therefore appears to be the major executioner caspase. Moreover, a recent report describing caspase-1-dependent activation of caspase-7, but not of caspase-3, in macrophages in response to microbial stimuli supports the idea of a non-redundant function for caspase-7 downstream of caspase-1 (21).Commercially available “caspase-specific” tetrapeptide substrates are widely used for specific caspase detection, but they display substantial promiscuity and cannot be used to monitor individual caspases in cells (22, 23). Detecting proteolysis by measuring the release of C-terminal fluorophores, such as 7-amino-4-methylcoumarin (amc), restricts the specificity of these peptide substrates to non-prime cleavage site residues, which may have hampered the identification of specific cleavage events. To address this limitation, a recently developed proteomics technique, called proteomic identification of protease cleavage sites, was used to map both non-prime and prime preferences for caspase-3 and -7 on a tryptic peptide library (9). However, no clear distinction in peptide recognition motifs between caspase-3 and -7 could be observed (9). Because not all classical caspase cleavage sites are processed (7), structural or post-translational higher order constraints are likely involved in steering the cleavage site selectivity. Peptide-based approaches generally overlook such aspects.We made use of the COFRADIC N-terminal peptide sorting methodology (24–26) to profile proteolytic events of caspase-3 and -7 in a macrophage proteome labeled by triple stable isotope labeling by amino acids in cell culture (SILAC), which allowed direct comparison of peak intensities in peptide MS spectra and consequent quantification of N termini that are equally, preferably, or exclusively generated by the action of caspase-3 or -7 (26, 27). We identified 55 cleavage sites in 48 protein substrates, encompassing mutual, preferred, and unique caspase-3 and -7 cleavage sites. 相似文献
18.
Benzylpenicillin acylase, which hydrolyzes benzylpenicillin to 6-aminopenicillanic acid, was found to be widely distributed among members of the Schizomycetes, particularly in gram-negative bacteria, and in the genus Nocardia. The hydrolysis of a series of biosynthetic and semisynthetic penicillins by freeze-dried cells of a strain of Nocardia and of Proteus was studied. Benzylpenicillin was the preferred substrate; all departures from the benzylpenicillin side-chain structure led to reduction of substrate activity (the greater the departure, the greater the reduction in activity). Penicillin amides and methyl esters were also hydrolyzed, as were suitable N-acyl derivatives of 7-aminocephalosporanic acid. Occurrence of an enzyme activity which hydrolyzes benzylpenicillinamide to benzylpenicillin was detected in certain strains of yeasts. 相似文献
19.
It has been shown that all selectins recognize the carbohydrate epitopes sialyl Lewisx and sialyl Lewisa. For the establishment of the structure-activity relationship, the efficient synthesis of these tetrasaccharides and derivatives is therefore of vital interest. The glycosyl transferase-mediated approach is summarized with emphasis on the use of modified acceptors and modified sugar-nucleotide donors. A survey of the involved enzymes: (1-3) and (1-4)galactosyl transferases, (2-3)sialyl transferase, FucT III and FucT VI reveals that the enzymatic synthesis is highly efficient for the rapid preparation of sialyl Lewisx- and sialyl Lewisa-derivatives. 相似文献