Role of lysine, tryptophan and calcium in the beta-elimination activity of a low-molecular-mass pectate lyase from Fusarium moniliformae.

An extracellular pectate lyase from Fusarium moniliformae was purified to homogeneity by affinity chromatography followed by gel filtration, with a yield of 76.5%. Laser desorption MS of the enzyme gave a molecular mass of 12,133.5 +/- 2.5 Da. The pectate lyase was a glycoprotein with a 5% carbohydrate content and had a pl value of 9.1. Atomic-emission spectrometry showed that Ca2+ was a part of the holoenzyme held by carboxy groups of the protein. These results support the hypothesis of a putative Ca2+ site suggested by Yodder, Keen and Jurnak [(1993) Science 260, 1503-1507] in the crystal structure of pectate lyase C of Erwinia chrysanthemi. Loss of Ca2+ was observed by treatment with EGTA or carboxy-modifying Woodward's reagent K, with subsequent loss of enzyme activity. Tryptophan fluorescence quenching showed that Ca2+ does not affect binding of substrate to enzyme. Chemical-modification and substrate-protection studies showed the presence of lysine and tryptophan at or near the active site of the pectate lyase. Chemically modified enzyme showed no major structural changes as determined by CD. Amino acid analyses of native, trinitrobenzenesulphonate (TBNS)-treated and substrate-protected TNBS-treated enzyme showed that a single essential residue of lysine is present at or near the active-site. Substrate-affinity studies showed that tryptophan could be essential for substrate binding, whereas lysine could be involved in the catalysis. Fluorescence quenching further confirmed the involvement of tryptophan in substrate binding. The reaction mechanism involving beta-elimination by this enzyme is discussed.

Implication of tryptophan and histidine in the active site of endo-polygalacturonase from Aspergillus ustus: elucidation of the reaction mechanism.

The mechanism of action for the hydrolysis of polygalacturonic acid by the enzyme endo-polygalacturonase (poly(1,4-alpha-D-galacturonide) glycanohydrolase, EC 3.2.1.15) was investigated. The enzyme from Aspergillus ustus was purified to homogeneity and used for the study. The endo-polygalacturonase had a molecular weight of 36,000 daltons, a pI of 8.3, specific activity of 785 units/mg, Km of 0.82 mg/ml, and Vmax of 976 micromoles of product min-1 mg-1. Amino acids involved in the catalysis were identified by chemical modification and the active site characterized. Inhibition by hydroxynitrobenzyl bromide and diethylpyrocarbonate, followed by substrate protection studies showed that tryptophan and histidine were involved at or near the active site. Kinetic constants of partially inhibited enzyme, suggest the involvement of tryptophan in substrate binding and histidine in catalysis. Quenching of tryptophan fluorescence of the enzyme in the presence of polygalacturonic acid substantiated the conclusion that tryptophan was involved in substrate binding. An isotope effect of 1.8 was observed with deuterated water on the Vmax of the endo-polygalacturonase, with the proton inventory giving a linear relationship. The proposed mechanism involves a single proton transfer from the histidine residue of the enzyme to the glycosidic oxygen and hydrolysis by the addition of a water molecule.

The two cysteine endopeptidases of legume seeds: purification and characterization by use of specific fluorometric assays.

Two endopeptidases are present in the seeds of Vigna aconitifolia (moth bean), and their activities increase during germination. One enzyme, which we term "vignain," can be assayed with benzyloxycarbonyl-phenylalanyl-arginyl-7-(4-methyl)coumarylamide as substrate. The second is legumain (EC 3.4.22.34), which can be assayed with benzyloxycarbonyl-alanyl-alanyl-asparaginyl-7-(4-methyl)-coumarylamide. The enzymes were purified, and their specificities for substrates and inhibitors were examined. Vignain has properties expected of a cysteine endopeptidase of the papain family, with the exception of a remarkably low reactivity with iodoacetate. Legumain is a very atypical cysteine endopeptidase, being insensitive to inhibition by chicken cystatin and E-64 (L-3-carboxy-2,3-trans-epoxypropionyl-leucyl-amido(4-guanidino )butane), and reacting more rapidly with iodoacetamide than with iodoacetate. We discuss our findings in relation to the literature on the proteolytic enzymes of legume seeds.

Salt-tolerant and thermostable alkaline protease from Bacillus subtilis NCIM no. 64.

The proteolytic activity produced by a Bacillus subtilis isolated from a hot spring was investigated. Maximum protease production was obtained after 38 h of fermentation. Effects of various carbon and nitrogen sources indicate the requirement of starch and bacteriological peptone to be the best inducers for maximum protease production. Requirement for phosphorus was very evident, and the protease was secreted over a wide range of pH 5-11. The partially purified enzyme was stable at 60 degrees C for 30 min. Calcium ions were effective in stabilizing the enzyme, especially at higher temperature. The enzyme was extremely salt tolerant and retained 100% activity in 5M NaCl over 96 h. The molecular weight of the purified enzymes as determined by SDS-PAGE was 28,000. The enzyme was completely inactivated by PMSF, but little affected by urea, sodium dodecyl sulfate, and sodium tripoly phosphate.

Clostripain: characterization of the active site.

In view of the probability that clostripain (EC 3.4.22.8) is fundamentally different in structure from other known cysteine endopeptidases, it was of interest to examine the characteristics of the active site. Z-Phe-Lys-CH2S(CH3)2 irreversibly and rapidly inactivated clostripain, and leupeptin was found to be the most potent reversible inhibitor yet reported for the enzyme. Clostripain was inhibited weakly by some protein inhibitors of serine endopeptidases, and required Ca2+ for stability and activity. Mg2+ and Sr2+ were ineffective. Rapid inactivation by diethylpyrocarbonate, reversed by hydroxylamine, indicated that histidine is essential for catalytic activity. Clostripain was more rapidly inactivated by iodoacetamide than by iodoacetate, with unique pH-dependences of reaction.

Affinity purification of the novel cysteine proteinase papaya proteinase IV, and papain from papaya latex.

A procedure is described for the purification of a previously undetected cysteine proteinase, which we have called papaya proteinase IV, from spray-dried latex of the papaya (Carica papaya) plant. The purification involves affinity chromatography on Gly-Phe-aminoacetonitrile linked to CH-Sepharose 4B, with elution by 2-hydroxyethyl disulphide at pH 4.5. The product thus obtained is a mixture of almost fully active papain and papay proteinase IV, which are then separated by cation-exchange chromatography. A preliminary characterization of papaya proteinase IV showed it to be very similar to chymopapain in both molecular size and charge. However, the new enzyme is immunologically distinct from the previously characterized cysteine proteinases of papaya latex. It also differs in its lack of activity against the synthetic substrates of the other papaya proteinases, in its narrow specificity against protein substrates and its lack of inhibition by chicken cystatin. Papaya proteinase IV is abundant, contributing almost 30% of the protein in spray-dried papaya latex, and contamination of chymopapain preparations with this enzyme may account for some of the previously reported heterogeneity of chymopapain.

Calpain inhibition by peptide epoxides.

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.

Cystatin-like cysteine proteinase inhibitors from human liver.

Cysteine proteinase inhibitor (CPI) forms from human liver were purified from the tissue homogenate by alkaline denaturation of cysteine proteinases with which they are complexed, acetone fractionation, affinity chromatography on S-carboxymethyl-papain-Sepharose and chromatofocusing. The multiple forms of CPI were shown immunologically to be forms of two proteins, referred to as CPI-A (comprising the forms of relatively acidic pI) and CPI-B (comprising the more basic forms). CPI-A and CPI-B are similar in their Mr of about 12400, considerable stability to pH2, pH11 and 80 degrees C, and tight-binding inhibition of papain, several related cysteine proteinases and dipeptidyl peptidase I. Ki values were determined for papain, human cathepsins B, H and L, and dipeptidyl peptidase I. The affinity of CPI-A for cathepsin B was about 10-fold greater than that of CPI-B, whereas CBI-B showed about 100-fold stronger inhibition of dipeptidyl peptidase I. For all the cysteine proteinases the liver inhibitors were somewhat less tight binding than cystatin. The resemblance of both CPI-A and CPI-B in several respects to egg-white cystatin is discussed. CPI-A seems to correspond to the epithelial inhibitor described previously, and CPI-B to the inhibitor from other cell types [Järvinen & Rinne (1982) Biochim. Biophys. Acta 708, 210-217].

Cystatin, a protein inhibitor of cysteine proteinases. Improved purification from egg white, characterization, and detection in chicken serum.

The protein from chicken egg white that inhibits cysteine proteinases, and has been named 'cystatin', was purified by ovomucin precipitation, affinity chromatography on carboxymethylpapain-Sepharose and chromatofocusing. The final purification step separated two major forms of the protein (pI 6.5 and 5.6), with a total recovery of about 20% from egg white. By use of affinity chromatography and immunodiffusion it was shown that the inhibitor is also present at low concentrations in the serum of male and female chickens. Tryptic peptide maps of the separated forms 1 and 2 of egg-white cystatin were closely similar, and each form had the N-terminal sequence Ser-Glx-Asx. The two forms showed complete immunological identity, and neither contained carbohydrate. Ki values for the inhibition of cysteine proteinases were as follows: papain (less than 1 X 10(-11)M), cathepsin B (8 X 10(-10)M), cathepsin H (about 2 X 10(-8)M) and cathepsin L (about 3 X 10(-12)M). Some other cysteine proteinases, and several non-cysteine proteinases, were found not to be significantly inhibited by cystatin. The inhibition of the exopeptidase dipeptidyl peptidase I by cystatin was confirmed and the Ki found to be 2 X 10(-10)M. Inhibitor complexes with active cysteine proteinases and the inactive derivatives formed by treatment with iodoacetate, E-64 [L-trans-epoxysuccinylleucylamido(4-guanidino)butane] and benzyloxycarbonylphenylalanylalanyldiazomethane were demonstrated by isoelectric focusing and cation-exchange chromatography. The complexes dissociated in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (with or without reduction) with no sign of fragmentation of the inhibitor. Cystatin was found not to contain a free thiol group, and there was no indication that disulphide exchange plays any part in the mechanism of inhibition.

Action of rat liver cathepsin L on collagen and other substrates.

1. It has been found that cathepsin L is very susceptible to loss of activity through autolysis. When this is prevented by purification and storage of the enzyme as its mercury derivative, preparations are obtained with higher specific activity than previously. 2. Active-site titration shows, however, that even the new purification method does not give preparations in which the enzyme is 100% active. 3. Benzyloxycarbonylphenylalanylarginine 7-(4-methyl)coumarylamide has been discovered to be a very sensitive substrate for cathepsin L. Like all other known substrates for cathepsin L, however, it is also cleaved by cathepsin B. 4. Cathepsin L degrades insoluble collagen at pH 3.5 over 5-fold faster than at pH 6.0. The specific activity at pH 3.5 is 5-10-fold higher than that of cathepsin B (rat or human) or bovine spleen cathepsin N ('collagenolytic cathepsin'). 5. Qualitatively, the action of cathepsin L on collagen is similar to that of cathepsins B and N, i.e. selective cleavage of terminal peptides leads to conversion of beta- and higher components mainly to alpha-chains.

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

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.

E-64 [L-trans-epoxysuccinyl-leucyl-amido(4-guanidino)butane] and related epoxides as inhibitors of cysteine proteinases.

1. E-64 [L-trans-epoxysuccinyl-leucyl-amido(4-guanidino)butane] was found to be an active site directed irreversible inhibitor of all of the cysteine proteinases tested except clostripain, and not to affect other enzymes. Thus it seems to be a powerful selective inhibitor for cysteine proteinases in mammalian systems. 2. The very high rate of inactivation of some cysteine proteinases, including cathepsins B and L, allows the molarity of active enzyme to be determined by stoichiometric titration with E-64. This incidentally calibrates the rate assay of the enzyme in terms of molar concentrations. 3. Measurement of rates of reaction of E-64 and a number of optically active analogues with cathepsins B, H and L has given some insight into structure-activity relationships. Cathepsin H is far less reactive than the other enzymes, and generally cathepsins B and L have reactivities rather similar to each other with the epoxide inhibitors.