Glycine flanked by hydrophobic bulky amino acid residues as minimal sequence for effective subtilisin catalysis.

The specificity of alkaline mesentericopeptidase (a proteinase closely related to subtilisin BPN') for the C-terminal moiety of the peptide substrate (Pi' specificity) has been studied in both hydrolysis and aminolysis reactions. N-Anthraniloylated peptide p-nitroanilides as fluorogenic substrates and amino acid or peptide derivatives as nucleophiles were used in the enzymic peptide hydrolysis and synthesis. Both hydrolysis and aminolysis kinetic data suggest a stringent specificity of mesentericopeptidase and related subtilisins to glycine as P1' residue and predilection for bulky hydrophobic P2' residues. A synergism in the action of S1' and S2'subsites has been observed. It appears that glycine flanked on both sides by hydrophobic bulky amino acid residues is the minimal amino acid sequence for an effective subtilisin catalysis.     

The purification and properties of yeast proteinase B from Candida albicans.

A serine proteinase (ycaB) from the yeast Candida albicans A.T.C.C. 10261 was purified to near homogeneity. The enzyme was almost indistinguishable from yeast proteinase B (EC 3.4.21.48), and an Mr of 30,000 for the proteinase was determined by SDS/polyacrylamide-gel electrophoresis. The initial site of hydrolysis of the oxidized B-chain of insulin, by the purified proteinase, was the Leu-Tyr peptide bond. The preferential degradation at this site, analysed further with N-blocked amino acid ester and amide substrates, demonstrated that the specificity of the proteinase is determined by an extended substrate-binding site, consisting of at least three subsites (S1, S2 and S'1). The best p-nitrophenyl ester substrates were benzyloxycarbonyl-Tyr p-nitrophenyl ester (kcat./Km 3,536,000 M-1 X S-1), benzyloxycarbonyl-Leu p-nitrophenyl ester (kcat./Km 2,250,000 M-1 X S-1) and benzyloxycarbonyl-Phe p-nitrophenyl ester (kcat./Km 1,000,000 M-1 X S-1) consistent with a preference for aliphatic or aromatic amino acids at subsite S1. The specificity for benzyloxycarbonyl-Tyr p-nitrophenyl ester probably reflects the binding of the p-nitrophenyl group in subsite S'1. The presence of S2 was demonstrated by comparison of the proteolytic coefficients (kcat./Km) for benzyloxycarbonyl-Ala p-nitrophenyl ester (825,000 M-1 X S-1) and t-butyloxycarbonyl-Ala p-nitrophenyl ester (333,000 M-1 X S-1). Cell-free extracts contain a heat-stable inhibitor of the proteinase.     

Substrate specificity of enzymes from Bacillus mesentericus

The proteolytic enzymes of the sporogenous Bacillus mesentericus strains 64 and 8 were tested for their ability to hydrolyse different protein substrates. The enzymes were isolated using affinity chromatography on bacillichine-silochrome, and eluted with 25% isopropanol in 0.05 M Tris-HCl buffer, pH 8.0-8.4, containing 0.01 M CaCl2. Casein, hemoglobin, elastin, albumin and synthetic peptides, Z-L-Ala-Ala-Leu-pNa and Z-L-Ala-Gly-Leu-pNa, were used as substrates. The activity of esterase was assayed in terms of indophenyl acetate cleavage. The proteinases were compared with terrilytin, a commercial preparation. The proteinase of strain 64 was active in the hydrolysis of casein, hemoglobin and elastin; its specificity was close to that of terrilytin. The proteinase of strain 8 differed from them in a higher thrombolytic and fibrinolytic activity, and had a high esterase activity.     

Re-formation of disulphide bonds in reduced antithrombin III.

The separation of pepsin isoenzymes 1, 2, 3 and 5 (gastricsin) in human gastric juice was effected by chromatography on Mono Q ion-exchanger, and slow-moving proteinase was purified to homogeneity by using a modified procedure incorporating a novel affinity-chromatography step. The pH-activity profiles of these enzymes with mucus glycoprotein and basement-membrane substrates were determined; the profiles for pepsin 2 were noticeably different, and, in general, the pH optima for the hydrolysis of basement membrane were more acidic. Pepsin 1 expressed larger specificity constants (kcat./Km) than pepsin 3 with a series of synthetic peptide substrates, reflecting greater binding (smaller Km) by pepsin 1. Inhibitor studies at pH 1.7 and 4.5 with a series of P2-substituted lactoyl-pepstatins implied that valine at position P2 was optimal for inhibiting pepsins 1, 2 and 3 but detrimental for pepsin 5, whereas lysine at position P2 was tolerated well by pepsin 5 but not by pepsins 1, 2 and 3. The potency of lactoyl-pepstatin with lysine at position P2 did not increase as a function of pH. P2-substituted lactoyl-pepstatins failed to show any inhibitory selectivity among pepsins 1, 2 and 3.     

Enzyme-substrate interactions in the hydrolysis of peptide substrates by thermitase, subtilisin BPN', and proteinase K

Peptide substrates of the general structure acetyl-Alan (n = 2-5), acetyl-Pro-Ala-Pro-Phe-Alan-NH2 (n = 0-3), and acetyl-Pro-Ala-Pro-Phe-AA-NH2 (AA = various amino acids) were synthesized and used to investigate the enzyme-substrate interactions of the microbial serine proteases thermitase, subtilisin BPN', and proteinase K on the C-terminal side of the scissile bond. The elongation of the substrate peptide chain up to the second amino acid on the C-terminal side (P'2) enhances the hydrolysis rate of thermitase and subtilisin BPN', whereas for proteinase K an additional interaction with the third amino acid (P'3) is possible. The enzyme subsite S'1 specificity of the proteases investigated is very similar. With respect to kcat/Km values small amino acid residues such as Ala and Gly are favored in this position. Bulky residues such as Phe and Leu were hydrolyzed to a lower extent. Proline in P'1 abolishes the hydrolysis of the substrates. Enzyme-substrate interactions on the C-terminal side of the scissile bond appear to affect kcat more than Km for all three enzymes.     

Design of selective substrates of proteinase 3 using combinatorial chemistry methods

In this study, chemical synthesis of the selective chromogenic/fluorogenic substrates for proteinase 3 is described. The substrates’ sequence was obtained using combinatorial chemistry methods. Deconvolution of the tripeptide library against proteinase 3 with general formula ABZ-X₃-X₂-X₁-ANB-NH₂ yielded the active sequence. Selected peptide was further modified on its C terminus to investigate the impact of chromophore moiety modification on enzyme-substrate interaction. To determine specificity, activity of selected substrates was characterized against proteinase 3 and neutrophil elastase. Finally, the peptide ABZ-Tyr-Tyr-Abu-ANB-NH₂ displayed the highest value of specificity constant (k_cat/K_M = 189 × 10³ M⁻¹ s⁻¹) for proteinase 3. To the best of our knowledge, this is the first short peptide that undergoes selective proteolysis by proteinase 3 and displays no significant hydrolysis in the presence of human neutrophil elastase and cathepsin G.     

Ananain: a novel cysteine proteinase found in pineapple stem

A previously unknown cysteine proteinase, named ananain, has been isolated from crude commercial pineapple stem bromelain. The purification procedure involved affinity chromatography on Sepharose-Gly-Phe-glycinaldehyde semicarbazone, and cation-exchange chromatography. The relative molecular mass of ananain was very similar to that of bromelain (25,000 and 26,000, respectively), but ananain differed greatly in specificity for hydrolysis of peptide and protein substrates. The new enzyme behaved as a typical cysteine proteinase in showing strong inhibition by chicken cystatin, whereas bromelain was scarcely affected. Ananain was also shown to be immunologically distinct from bromelain. The significance of the discovery of ananain for the interpretation of previous work on "bromelain" is pointed out.     

Leu254 residue and calcium ions as new structural determinants of carboxypeptidase T substrate specificity

New determinants of Thermoactinomyces vulgaris carboxypeptidase T (CPT) substrate specificity--structural calcium ions and Leu254 residue--were found by means of steady-state kinetics and site-directed mutagenesis. The removal of calcium ions shifted the selectivity profile of hydrolysis of tripeptide substrates with C-terminal Leu, Glu, and Arg from 64/1.7/1 to 162/1.3/1. Substitution of the hydrophobic Leu254 in CPT for polar Asn did not change hydrolysis efficiency of substrates with C-terminal Leu and Arg, but resulted in more than 28-fold decrease in activity towards the substrate with C-terminal Glu. It is shown that the His68 residue is not a structural determinant of CPT specificity.     

Selective cleavage of glycyl bonds by papaya proteinase IV

The specificity of papaya proteinase IV (PPIV) has been examined with small substrates and a protein. With both classes of substrate, the enzyme shows a marked selectivity for cleaving glycyl bonds. Boc-Ala-Ala-Gly-NHPhNO₂ is a convenient substrate for routine assays that discriminate well against chymopapain, the most common contaminant of PPIV. Sixteen cleavage points in β-trypsin were identified, of which 13 are glycyl bonds. Tentative suggestions are made as to the reasons for lack of cleavage of some other glycyl bonds. The structure of PPIV has been modelled on that of papain, and we suggest that the replacement of the highly conserved residues Gly-65 and Gly-23 by arginine and glutamic acid, respectively, can account for the specificity of PPIV.     

Purification and characterization of a protein inhibitor of the 20S proteasome (macropain).

An inhibitory protein for the 20S proteasome (also known as macropain, the multicatalytic proteinase complex and 20S proteinase) has been purified from bovine red blood cells. The inhibitor has an apparent molecular weight of 31,000 on SDS-PAGE and appears to form multimers under nondenaturing conditions. This protein inhibited all three of the putatively distinct catalytic activities of proteasome A (the active form of the proteinase) characterized by the hydrolysis of synthetic peptides such as Z-VLR-MNA, Z-GGL-AMC or Suc-LLVY-AMC and Z-LLE-beta NA. The inhibitor also prevented the hydrolysis of large protein substrates such as casein, lysozyme and bovine serum albumin. Proteasome L (the latent form of the proteinase) does not degrade these large protein substrates, but does hydrolyze the three synthetic peptides at rates similar to those by proteasome A. The inhibitor inhibited only two of these peptidase activities of proteasome L (hydrolysis of Z-GGL-AMC and of Z-LLE-beta NA or Suc-LLVY-AMC); it had no effect on the hydrolysis of Z-VLR-MNA. The inhibitor was specific for inhibition of the proteasome and had no effect on the activity of any other proteinase tested including trypsin, chymotrypsin, papain, subtilisin and both isoforms of calpain. Kinetic analysis indicates that the inhibitor interacted with the proteasome by a mechanism involving tight-binding. Because the proteasome appears to be a key component of the ATP/ubiquitin-dependent pathway of intracellular protein degradation, the inhibitor may represent an important regulatory protein of this pathway.     

Some Characteristics of Hydrolysis of Synthetic Substrates and Proteins by the Alkaline Proteinase from Aspergillus sojae

The specificity of the alkaline proteinase from Aspergillus sojae was investigated. In the specificity studies with synthetic substrates, the enzyme hydrolyzed the peptide linkages involving the carboxyl group of leucine, tyrosine, phenylalanine, arginine and lysine. In the hydrolysis of natural proteins, the enzyme liberated relatively large peptides and traces of free amino acids, suggesting that the enzyme is of a typical endo-type.

N- and C-Terminal amino acid residues appearing during time course digestion of various proteins were determined. Considering the influence of amino acid composition of substrates on the frequencies of appearance of the terminal amino acids, it was estimated that the susceptibility of peptide bonds of substrate to the enzyme depends mainly on the carboxyl side residues, and, to far less extent, on the amino side residues of the peptide bonds. The enzyme showed relatively high specificity for lysine, tyrosine, histidine, arginine and phenylalanine residues at the carboxyl side of the susceptible linkages.     

Stabilization from autoproteolysis and kinetic characterization of the human T-cell leukemia virus type 1 proteinase

We have developed a system for expression and purification of wild-type human T-cell leukemia virus type 1 (HTLV-1) proteinase to attain sufficient quantities for structural, kinetic, and biophysical investigations. However, similar to the human immunodeficiency virus type 1 (HIV-1) proteinase, HTLV-1 proteinase also undergoes autoproteolysis rapidly upon renaturation to produce two products. The site of this autoproteolytic cleavage was mapped, and a resistant HTLV-1 proteinase construct (L40I) as well as another construct, wherein the two cysteine residues were exchanged to alanines, were expressed and purified. Oligopeptide substrates representing the naturally occurring cleavage sites in HTLV-1 were good substrates of the HTLV-1 proteinase. The kinetic parameters kcat and Km were nearly identical for all the three enzymes. Although three of four peptides representing HTLV-1 proteinase cleavage sites were fairly good substrates of HIV-1 proteinase, only two of nine peptides representing HIV-1 proteinase cleavage sites were hydrolyzed by the HTLV-1 proteinase, suggesting substantial differences in the specificity of the two enzymes. The large difference in the specificity of the two enzymes was also demonstrated by inhibition studies. Of the several inhibitors of HIV-1 or other retroviral proteinases that were tested on HTLV-1 proteinase, only two inhibit the enzyme with a Ki lower than 100 nM.     

New subtilisin-like collagenase from leaves of common plantain

A new subtilisin-like proteinase hydrolyzing chromogenic peptide substrate Glp-Ala-Ala-Leu-p-nitroanilide optimally at pH 8.1 was found in common plantain leaves. The protease named plantagolisin was isolated by ammonium sulfate precipitation of the leaves' extract followed by affinity chromatography on bacitracin-Sepharose and ion-exchange chromatography on Mono Q in FPLC regime. Its molecular mass is 19000 Da and pI 5.0. pH-stability range is 7-10 in the presence of 2 mM Ca(2+), temperature optimum is 40 degrees C. The substrate specificity of subtilase towards synthetic peptides and insulin B-chain is comparable with that of two other subtilisin-like serine proteinases: proteinase from leaves of the sunflower and taraxalisin. Besides, the proteinase is able to hydrolyze substrates with Pro in P(1) position. The enzyme hydrolyzes collagen. alpha and beta chains are hydrolyzed simultaneously in parallel; there are only low-molecular-mass hydrolysis products in the sample after 2 h of incubation. Pure serine proteinase was inactivated by specific serine proteinases inhibitors: diisopropylfluorophosphate, phenylmethylsulfonyl fluoride and Hg(2+). The plantagolisin N-terminal sequence ESNSEQETQTESGPGTAFL-, traced for 19 residues, revealed 37% homology with that of subtilisin from yeast Schizosaccharomyces pombe.     

The pH dependence of the hydrolysis of chromogenic substrates of the type, Lys-Pro-Xaa-Yaa-Phe-(NO2)Phe-Arg-Leu, by selected aspartic proteinases: evidence for specific interactions in subsites S3 and S2

Variation in the kinetic parameters, kcat and Km, with pH has been used to obtain evidence for significant acid-dissociation processes in the hydrolysis of octapeptide substrates by three aspartic proteinases. These substrates are all cleaved at the peptide bond between a Phe (P1) and a p-nitroPhe (P1') residue resulting in a shift in absorbance at 300 nm that facilitates kinetic measurements. The substrates differ in the amino-acid residues present in the P3 and the P2 positions. Porcine pepsin, calf chymosin, and the aspartic proteinase from Endothia parasitica all show pH dependencies that imply that favorable or unfavorable interactions can occur with the S3 or S2 areas of the enzyme-active site. Examination of the crystallographically determined structure of the E. parasitica proteinase and consideration of the amino-acid sequence differences between the three enzymes suggests that the origin of the pH effects arises from favorable interactions between Glu-13 (COO-) of pig pepsin and Thr (OH) or His (ImH+) in P3 of a substrate. Similarly, Lys-220 (NH3+) of chymosin and a Glu (COO-) in P2 of a substrate may produce a favorable interaction and Asp-77 (COO-) of E. parasitica proteinase and a Glu (COO-) in P2 of a substrate may produce an unfavorable interaction. These results lead to possible explanations for subtle specificity differences within a family of homologous enzymes, and suggest loci for study by site-directed mutagenesis.     

New class of sensitive and selective fluorogenic substrates for serine proteinases. Amino acid and dipeptide derivatives of rhodamine.

A series of dipeptide derivatives of Rhodamine, each containing an arginine residue in the P1 position and one of ten representative benzyloxycarbonyl (Cbz)-blocked amino acids in the P2 position, has been synthesized, purified and characterized as substrates for serine proteinases. These substrates are easily prepared with high yields. Cleavage of a single amide bond converts the non-fluorescent bisamide substrate into a highly fluorescent monoamide product. Macroscopic kinetic constants for the interaction of these substrates with bovine trypsin, human and dog plasmin, and human thrombin are reported. Certain of these substrates exhibit extremely large specificity constants. For example, the kcat./Km for bovine trypsin with bis-(N-benzyloxycarbonylglycyl-argininamido)-Rhodamine [(Cbz-Gly-Arg-NH)2-Rhodamine] is 1 670 000 M-1 X S-1. Certain of these substrates are also highly selective. For example, the most specific substrate for human plasmin, (Cbz-Phe-Arg-NH2)-Rhodamine, is not hydrolysed by human thrombin, and the most specific substrate for human thrombin, (Cbz-Pro-Arg-NH)2-Rhodamine, is one of the least specific substrates for human plasmin. Comparison of the kinetic constants for hydrolysis of the dipeptide substrates with that of the single amino acid derivative, (Cbz-Arg-NH)2-Rhodamine, indicates that selection of the proper amino acid residue in the P2 position can effect large increases in substrate specificity. This occurs primarily as a result of an increase in kcat. as opposed to a decrease in Km and, in certain cases, is accompanied by a large increase in selectivity. Because of their high degree of sensitivity and selectivity, these Rhodamine-based dipeptide compounds should be extremely useful substrates for studying serine proteinases.     

Specificity of Proteinase K from Tritirachium album Limber for Synthetic Peptides

The specificity of proteinase K from Tritirachium album Limber was determined using various synthetic peptide substrates. The esterase activity against N-acylated amino acid esters indicated that the enzyme is primarily specific against aromatic or hydrophobic amino acid residues at the carboxyl side of the splitting point. Secondary interaction for hydrolysis was also studied using peptide esters or others, which showed that the enzyme activity is markedly promoted by elongating the peptide chain to the N-terminal from the splitting point. Thus, peptide chloromethyl ketone derivatives such as Cbz-Ala-Gly-PheCH₂Cl inactivated the enzyme activity markedly.     

Bilayer permeability-based substrate selectivity of an enzyme in liposomes

Liposomes were prepared from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), which contained the water soluble proteinase alpha-chymotrypsin. This liposome entrapped enzyme showed selectivity for externally added substrates in that only small substrates (benzoyl-l-Tyr-p-nitroanilide or acetyl-l-Phe-p-nitro-anilide)-for which the liposome bilayer was permeable-were transformed into products. Large substrates (succinyl-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide or casein) could not penetrate from the external aqueous phase into the liposomes, and were not hydrolyzed. This substrate selectivity is entirely based on the compartimentation and permeability properties of the liposome microreactor.     

A consensus sequence for substrate hydrolysis by rhinovirus 3C proteinase

Kinetic constants were determined for the hydrolysis of a series of synthetic peptide substrates by recombinant rhinovirus (HRV 14) 3C proteinase. Systematic removal or replacement of individual residues indicated that the minimum sequence required for effective cleavage by the viral cysteine proteinase was P5-Val/Thr-P3-P2-Gln-Gly-Pro.     

Evolutionary optimization of peptide substrates for proteases that exhibit rapid hydrolysis kinetics

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.     

Characterisation of cysteine proteinases responsible for digestive proteolysis in guts of larval western corn rootworm (Diabrotica virgifera) by expression in the yeast Pichia pastoris

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.