scholarly journals Calpain inhibition by peptide epoxides

1985 ◽  
Vol 230 (2) ◽  
pp. 509-516 ◽  
Author(s):  
C Parkes ◽  
A A Kembhavi ◽  
A J Barrett
Keyword(s):  
Smooth Muscle ◽  
Active Site ◽  
Rate Constants ◽  
Cysteine Proteinase ◽  
Limited Proteolysis ◽  
Enzymic Activity ◽  
Isoelectric Precipitation ◽  
Structural Analogues ◽  
Calpain Inhibition ◽  
Calpain Ii

A Ca2+-activated cysteine proteinase (calpain II) was purified from chicken gizzard smooth muscle by use of isoelectric precipitation, (NH4)2SO4 fractionation, chromatography on DEAE-Sepharose CL-6B, Reactive-Red 120-agarose and Mono Q. The apparent second-order rate constants for the inactivation of calpain by a series of structural analogues of L-3-carboxy-trans-2, 3-epoxypropionyl-leucylamido-(4-guanidino)butane (E-64) were determined. The fastest rate of inactivation was observed with L-3-carboxy-trans-2, 3-epoxypropionyl-leucylamido-(4-benzyloxy-carbonylamino)buta ne. It was possible to determine the active-site molarity of solutions of calpain by titration with E-64. When incubated with Ca2+, calpain underwent several steps of intermolecular limited proteolysis, via multiple pathways, followed by a slower loss of enzymic activity. The proteolytic steps preceding the loss of activity did not affect the rates of reaction of calpain with E-64 analogues.

scholarly journals Characterization of proteoglycan degradation by calpain

1992 ◽  
Vol 285 (3) ◽  
pp. 857-862 ◽  
Author(s):  
K Suzuki ◽  
K Shimizu ◽  
T Hamamoto ◽  
Y Nakagawa ◽  
T Murachi ◽  
...  
Keyword(s):  
Core Protein ◽  
Cysteine Proteinase ◽  
Limited Proteolysis ◽  
Side Chains ◽  
Proteoglycan Degradation ◽  
Cartilage Proteoglycans ◽  
Neutral Conditions ◽  
Calpain Ii ◽  
Enzyme Ratio

Degradation of cartilage proteoglycans was investigated under neutral conditions (pH 7.5) by using pig kidney calpain II (EC 3.4.22.17; Ca(2+)-dependent cysteine proteinase). Aggregate and monomer degradation reached a maximum in 5 min at 30 degrees C when the substrate/enzyme ratio was less than 1000:1. The mode of degradation was limited proteolysis of the core protein; the size of the products was larger than that of papain-digested products and comparable with that of trypsin-digested products. The hyaluronic acid-binding region was lost from the major glycosaminoglycan-bearing region after incubation with calpain II. Calpains thus may affect the form of proteoglycans in connective tissue. Ca(2+)-dependent proteoglycan degradation was unique in that proteoglycans adsorb large amounts of Ca2+ ions rapidly before activation of calpain II: 1 mg of pig cartilage proteoglycan monomer adsorbed 1.3-1.6 mu equiv. of Ca2+ ions before activation of calpain II, which corresponds to half the sum of anion groups in glycosaminoglycan side chains. This adsorption of Ca2+ was lost after solvolysis of proteoglycan monomer with methanol/50 mM-HCl, which was used to desulphate glycosaminoglycans. Therefore cartilage proteoglycans are not merely the substrates of proteolysis, but they may regulate the activation of Ca(2+)-dependent enzymes including calpains through tight chelation of Ca2+ ions between glycosaminoglycan side chains.

scholarly journals L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L

1982 ◽  
Vol 201 (1) ◽  
pp. 189-198 ◽  
Author(s):  
A J Barrett ◽  
A A Kembhavi ◽  
M A Brown ◽  
H Kirschke ◽  
C G Knight ◽  
...  
Keyword(s):  
Active Site ◽  
Cathepsin B ◽  
Cysteine Proteinase ◽  
Cathepsin L ◽  
Competitive Inhibitor ◽  
Serine Proteinases ◽  
Cysteine Proteinases ◽  
Leucocyte Elastase ◽  
Structural Analogues ◽  
Stoichiometric Reaction

1. L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) at a concentration of 0.5 mM had no effect on the serine proteinases plasma kallikrein and leucocyte elastase or the metalloproteinases thermolysin and clostridial collagenase. In contrast, 10 muM-E-64 rapidly inactivated the cysteine proteinases cathepsins B, H and L and papain (t0.5 = 0.1-17.3s). The streptococcal cysteine proteinase reacted much more slowly, and there was no irreversible inactivation of clostripain. The cysteine-dependent exopeptidase dipeptidyl peptidase I was very slowly inactivated by E-64. 2. the active-site-directed nature of the interaction of cathepsin B and papain with E-64 was established by protection of the enzyme in the presence of the reversible competitive inhibitor leupeptin and by the stereospecificity for inhibition by the L as opposed to the D compound. 3. It was shown that the rapid stoichiometric reaction of the cysteine proteinases related to papain can be used to determine the operational molarity of solutions of the enzymes and thus to calibrate rate assays. 4. The apparent second-order rate constants for the inactivation of human cathepsins B and H and rat cathepsin L by a series of structural analogues of E-64 are reported, and compared with those for some other active-site-directed inhibitors of cysteine proteinases. 5. L-trans-Epoxysuccinyl-leucylamido(3-methyl)butane (Ep-475) was found to inhibit cathepsins B and L more rapidly than E-64. 6. Fumaryl-leucylamido(3-methyl)butane (Dc-11) was 100-fold less reactive than the corresponding epoxide, but was nevertheless about as effective as iodoacetate.

scholarly journals The effects of autolysis on the structure of chicken calpain II

1987 ◽  
Vol 248 (2) ◽  
pp. 579-588 ◽  
Author(s):  
C Crawford ◽  
A C Willis ◽  
J Gagnon
Keyword(s):  
Structural Changes ◽  
Structural Integrity ◽  
Large Subunit ◽  
Limited Proteolysis ◽  
Gel Permeation Chromatography ◽  
Enzymic Activity ◽  
Amino Acid Sequences ◽  
Cleavage Sites ◽  
Terminal Amino ◽  
Calpain Ii

When chicken calpain II autolysed in the presence of Ca2+, it underwent limited proteolysis to give peptides of Mr 54,000 and 37,000, and several of Mr approx. 30,000 and 18,000. The autolytic peptides were purified and their N-terminal amino acid sequences determined. By comparison of these sequences with the known sequence of the complete calpain molecule, the autolytic cleavage sites were identified. The structural integrity of the molecule during autolysis was investigated by gel-permeation chromatography. Experiments were also done to test the reversibility of adding EDTA to calpain during autolysis, measured as recoverable enzyme activity assayed in the presence of Ca2+. The results are presented in terms of a model for the structural changes occurring in calpain during autolysis. It was concluded that the loss of enzymic activity, which is a consequence of autolysis, was due to dissociation of the autolytic peptides after cleavage of the calpain large subunit within the third domain.

The structure and mechanism of action of papain

1970 ◽  
Vol 257 (813) ◽  
pp. 237-248 ◽  
Keyword(s):  
Mechanism Of Action ◽  
Catalytic Mechanism ◽  
Proteolytic Enzymes ◽  
Cysteine Proteinase ◽  
Preceding Paper ◽  
Cysteine Residue ◽  
Enzymic Activity ◽  
Critical Survey ◽  
Cysteine Proteinases ◽  
Stem Bromelain

The cysteine proteinases form a group of enzymes which depend for their enzymic activity on the thiol group of a cysteine residue. Several which occur in plants have been investigated extensively and include papain, ficin and stem bromelain (Smith & Kimmel i960). Although the term papain, introduced last century to describe the proteolytic principle in papaya latex (Wurtz & Bouchut 1879) is still used to describe crude dried latex, the crystalline enzyme is readily obtained (Kimmel & Smith 1954). Ficin is known to consist of several closely related enzymes which have been resolved (Sgarbieri, Gupte, Kramer & Whitaker 1964), but for most structural and mechanistic studies the unresolved mixture of enzymes has been used. Stem bromelain also appears to be a mixture of at least two proteolytic enzymes which have not yet been resolved (Ota, Moore & Stein 1962; Murachi 1964). In spite of the recognized heterogeneity of ficin and stem bromelain, it does seem that both structurally and mechanistically they are similar to papain. Only one bacterial cysteine proteinase has received a detailed study, namely, streptococcal proteinase, and it appears to have little or no relation in its amino acid sequence with the plant enzymes (Liu, Stein, Moore & Elliott 1965). The functional groups involved in the catalytic mechanism are apparently the same as in the plant proteinases (Gerwin, Stein & Moore 1966; Liu 1967; Husain & Lowe 1968 a , c ), but the mechanism of action has not been extensively studied. It may well be however that the plant and bacterial cysteine proteinases have converged onto a similar mechanism of action by two independent evolutionary pathways, as now seems apparent for the animal and bacterial serine proteinases (Alden, Wright & Kraut, this volume, p. 119). Because the tertiary crystal structure of papain (Drenth, Jansonius, Koekoek, Swen & Wolthers 1968; see also the preceding paper, p. 231) is now known, a critical survey of this enzyme is apposite.

scholarly journals Avian reovirus core protein μA expressed in Escherichia coli possesses both NTPase and RTPase activities

2007 ◽  
Vol 88 (6) ◽  
pp. 1797-1805 ◽  
Author(s):  
Yu Pin Su ◽  
Jui Huang Shien ◽  
Hung Jen Liu ◽  
Hsien Sheng Yin ◽  
Long Huw Lee
Keyword(s):  
Active Site ◽  
Rna Synthesis ◽  
Core Protein ◽  
Divalent Cations ◽  
Fusion Peptide ◽  
Enzymic Activity ◽  
Avian Reovirus ◽  
Phosphate Release ◽  
N Terminus ◽  
Rna Triphosphatase

Analysis of the amino acid sequence of core protein μA of avian reovirus has indicated that it may share similar functions to protein μ2 of mammalian reovirus. Since μ2 displayed both nucleotide triphosphatase (NTPase) and RNA triphosphatase (RTPase) activities, the purified recombinant μA ( μA) was designed and used to test these activities. μA was thus expressed in bacteria with a 4.5 kDa fusion peptide and six His tags at its N terminus. Results indicated that  μA possessed NTPase activity that enabled the protein to hydrolyse the β–γ phosphoanhydride bond of all four NTPs, since NDPs were the only radiolabelled products observed. The substrate preference was ATP>CTP>GTP>UTP, based on the estimated k cat values. Alanine substitutions for lysines 408 and 412 (K408A/K412A) in a putative nucleotide-binding site of  μA abolished NTPase activity, further suggesting that NTPase activity is attributable to protein  μA. The activity of  μA is dependent on the divalent cations Mg2+ or Mn2+, but not Ca2+ or Zn2+. Optimal NTPase activity of  μA was achieved between pH 5.5 and 6.0. In addition,  μA enzymic activity increased with temperature up to 40 °C and was almost totally inhibited at temperatures higher than 55 °C. Tests of phosphate release from RNA substrates with  μA or K408A/K412A  μA indicated that  μA, but not K408A/K412A  μA, displayed RTPase activity. The results suggested that both NTPase and RTPase activities of  μA might be carried out at the same active site, and that protein μA could play important roles during viral RNA synthesis.

scholarly journals Ligand binding on to maize (Zea mays) malate synthase: a structural study

1994 ◽  
Vol 303 (2) ◽  
pp. 413-421 ◽  
Author(s):  
S Beeckmans ◽  
A S Khan ◽  
L Kanarek ◽  
E Van Driessche
Keyword(s):  
Zea Mays ◽  
Ligand Binding ◽  
Conformational Change ◽  
Limited Proteolysis ◽  
Enzymic Activity ◽  
Malate Synthase ◽  
Acetyl Coa ◽  
Original Activity ◽  
Kinetic Measurements ◽  
Michaelis Constants

A kinetic and ligand binding study on maize (Zea mays) malate synthase is presented. It is concluded from kinetic measurements that the enzyme proceeds through a ternary-complex mechanism. Michaelis constants (Km,glyoxylate and Km,acetyl-CoA) were determined to be 104 microM and 20 microM respectively. C.d. measurements in the near u.v.-region indicate that a conformational change is induced in the enzyme by its substrate, glyoxylate. From these studies we are able to calculate the affinity for the substrate (Kd,glyoxylate) as 100 microM. A number of inhibitors apparently trigger the same conformational change in the enzyme, i.e. pyruvate, glycollate and fluoroacetate. Another series of inhibitors bearing more bulky groups and/or an extra carboxylic acid also induce a conformational change, which is, however, clearly different from the former one. Limited proteolysis with trypsin results in cleavage of malate synthase into two fragments of respectively 45 and 19 kDa. Even when no more intact malate synthase chains are present, the final enzymic activity still amounts to 30% of the original activity. If trypsinolysis is performed in the presence of acetyl-CoA, the cleavage reaction is appreciably slowed down. The dissociation constant for acetyl-CoA (Kd,acetyl-CoA) was calculated to be 14.8 microM when the glyoxylate subsite is fully occupied by pyruvate and 950 microM (= 50 x Km) when the second subsite is empty. It is concluded that malate synthase follows a compulsory-order mechanism, glyoxylate being the first-binding substrate. Glyoxylate triggers a conformational change in the enzyme and, as a consequence, the correctly shaped binding site for acetyl-CoA is created. Demetallization of malate synthase has no effect on the c.d. spectrum in the near u.v.-region. Moreover, glyoxylate induces the same spectral change in the absence of Mg2+ as in its presence. Nevertheless, malate synthase shows no activity in the absence of the cation. We conclude that Mg2+ is essential for catalysis, rather than for the structure of the enzyme's catalytic site.

scholarly journals The two cathepsin B-like proteases of Arabidopsis thaliana are closely related enzymes with discrete endopeptidase and carboxydipeptidase activities

2018 ◽  
Vol 399 (10) ◽  
pp. 1223-1235 ◽  
Author(s):  
Andreas Porodko ◽  
Ana Cirnski ◽  
Drazen Petrov ◽  
Teresa Raab ◽  
Melanie Paireder ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Active Site ◽  
Cathepsin B ◽  
Cysteine Proteinase ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Substrate Specificities ◽  
Active Site Cleft ◽  
Molecular Modeling Studies ◽  
Human Cathepsin

Abstract The genome of the model plant Arabidopsis thaliana encodes three paralogues of the papain-like cysteine proteinase cathepsin B (AtCathB1, AtCathB2 and AtCathB3), whose individual functions are still largely unknown. Here we show that a mutated splice site causes severe truncations of the AtCathB1 polypeptide, rendering it catalytically incompetent. By contrast, AtCathB2 and AtCathB3 are effective proteases which display comparable hydrolytic properties and share most of their substrate specificities. Site-directed mutagenesis experiments demonstrated that a single amino acid substitution (Gly336→Glu) is sufficient to confer AtCathB2 with the capacity to tolerate arginine in its specificity-determining S2 subsite, which is otherwise a hallmark of AtCathB3-mediated cleavages. A degradomics approach utilizing proteome-derived peptide libraries revealed that both enzymes are capable of acting as endopeptidases and exopeptidases, releasing dipeptides from the C-termini of substrates. Mutation of the carboxydipeptidase determinant His207 also affected the activity of AtCathB2 towards non-exopeptidase substrates, highlighting mechanistic differences between plant and human cathepsin B. This was also noted in molecular modeling studies which indicate that the occluding loop defining the dual enzymatic character of cathepsin B does not obstruct the active-site cleft of AtCathB2 to the same extent as in its mammalian orthologues.

scholarly journals Beyond the Active Site: Mechanistic Investigations of the Role of the Secondary Coordination Sphere and Beyond on Multi-Electron Electrocatalytic Reactions and Their Relations Between Kinetics and Thermodynamics

2020 ◽  
Author(s):  
Vincent Wang
Keyword(s):  
Thermodynamic Parameters ◽  
Coordination Sphere ◽  
Active Site ◽  
Rate Constants ◽  
Reduction Reaction ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Secondary Coordination Sphere ◽  
Catalytic Rate ◽  
Rate Limiting

<p>The development of an electrocatalyst with a rapid turnover frequency, low overpotential and long-term stability is highly desired for fuel-forming reactions, such as water splitting and CO<sub>2</sub> reduction. The findings of the scaling relationships between the catalytic rate and thermodynamic parameters over a wide range of electrocatalysts in homogeneous and heterogeneous systems provide useful guidelines and predictions for designing better catalysts for those redox reactions. However, such relationships also suggest that a catalyst with a high catalytic rate is typically associated with a high overpotential for a given reaction. Inspired by enzymes, the introduction of additional interactions through the secondary coordination sphere beyond the active site, such as hydrogen-bonding or electrostatic interactions, have been shown to offer a promising avenue to disrupt these unfavorable relationships. Herein, we further investigate the influence of these cooperative interactions on the faster chemical steps, in addition to the rate-limiting step widely examined before, for molecular electrocatalysts with the structural and electronic modifications designed to facilitate the dioxygen reduction reaction, CO<sub>2</sub> reduction reaction and hydrogen evolving reaction. Based on the electrocatalytic kinetic analysis, the rate constants for faster chemical steps and their correlation with the corresponding thermodynamic parameters are evaluated. The results suggest that the effects of the secondary coordination sphere and beyond on these fuel-forming reactions are not necessarily beneficial for promoting all chemical steps and no apparent relation between rate constants and thermodynamic parameters are found in some cases studied here, which may implicate the design of electrocatalysts in the future. Finally, these analyses demonstrate that the characteristic features for voltammograms and foot-of-the-wave-analysis plots are associated with the specific kinetic phenomenon among these multi-electron electrocatalytic reactions, which provides a useful framework to probe the insights of chemical and electronic modifications on the catalytic steps quantitatively (i.e. kinetic rate constants) and to optimize some of critical steps beyond the rate-limiting step.</p>

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