Proteázy obecně

Aminopeptidázy

• Free N terminus of the polypeptide chain
• Liberate a single amino acid residue, a dipeptide, or a tripeptide
• Remove the N-terminal Met
• May be found in heterologously expressed proteins
• Not in many naturally occurring mature proteins [11]
• Occur in a wide variety of microbial species
• Bacteria and fungi [11]
• Intracellular enzymes
• Single report on an extracellular aminopeptidase
• Produced by A. oryzae [11]
• Aminopeptidase I from Escherichia coli
• Large protease (400,000 Da)
• Broad pH optimum of 7.5 to 10.5
• Requires Mg2+ or Mn2+ for optimal activity [11]
• Bacillus licheniformis aminopeptidase
• Molecular weight of 34,000
• 1 g-atom of Zn2+ per mol
• Activity is enhanced by Co2+ ions [11]
• Aminopeptidase II from B. stearothermophilus
• Dimer with a molecular weight of 80,000 to 100,000
• Activated by Zn2+, Mn2+, or Co2+ ions [11]

Carboxypeptidases

• At C terminals of the polypeptide chain
• Liberate a single amino acid or a dipeptide
• Three major groups
• serine carboxypeptidases
• Metallocarboxypeptidases
• Cysteine carboxypeptidases
• serine carboxypeptidases
• From Penicillium spp., Saccharomyces spp., and Aspergillus spp.
• Similar in their substrate specificities
• Differ slightly in other properties such as
• PH optimum
• Stability
• Molecular weight
• Effect of inhibitors [11]
• Metallocarboxypeptidases from Saccharomyces spp. and Pseudomonas spp.
• Require Zn2+ or Co2+ for their activity
• Can also hydrolyze the peptides in which the peptidyl group is replaced by a pteroyl moiety or by acyl groups [11]

Endopeptidases

• Preferential action at the peptide bonds in the inner regions
• Presence of the free amino or carboxyl group
• Negative influence on enzyme activity

4 subgroups

• serine proteases
• Aspartic proteases
• Cysteine proteases
• Metalloproteases

• Rawlings and Barrett
• Code letter denoting the catalytic type
• S, C, A, M, or U (see above) followed by an artibrarily assigned number [11]

Serine proteases

• Presence of a serine group in their active site
• Viruses, bacteria, and eukaryotes
• In the exopeptidase, endopeptidase, oligopeptidase, and omega peptidase groups
• 20 families
• Further subdivided into about 6 clans with common ancestors
• The primary structures of the members of 4 clans are totally unrelated
• Chymotrypsin (SA)
• Subtilisin (SB)
• Carboxypeptidase C (SC)
• Escherichia d-Ala–d-Ala peptidase A (SE)
• At least four separate evolutionary origins for serine proteases
• Irreversible inhibition by:
• 3,4-dichloroisocoumarin (3,4-DCI)
• L-3-carboxytrans 2,3-epoxypropyl-leucylamido (4-guanidine) butane (E.64)
• Diisopropylfluorophosphate (DFP)
• Phenylmethylsulfonyl fluoride (PMSF)
• Tosyl-l-lysine chloromethyl ketone (TLCK)
• Some inhibited by
• Thiol reagents
• P-chloromercuribenzoate (PCMB)
• Generally active at neutral and alkaline pH
• Optimum between pH 7 and 11
• Broad substrate specificities
• Esterolytic
• Amidase activity [11]
• Molecular masses range between 18 and 35 kDa
• Isoelectric points between pH 4 and 6
• Serine alkaline proteases
• Active at highly alkaline pH represent the largest subgroup [11]

Serine alkaline proteases

• Produced by several bacteria, molds, yeasts, and fungi
• Arthrobacter
• Streptomyces
• Flavobacterium spp.
• Bacillus spp.
• Subtilisins are the best known [11]
• S. cerevisiae
• Filamentous fungi such as Conidiobolus spp.
• Aspergillus
• Neurospora spp. [11]
• Inhibited by
• DFP
• Potato protease inhibitor
• Not inhibited by
• Tosyl-l-phenylalanine chloromethyl ketone (TPCK)
• TLCK
• Substrate specificity
• Similar to but less stringent than that of chymotrypsin
• Hydrolyze a peptide bond which has tyrosine, phenylalanine, or leucine at the carboxyl side
• Optimal pH of cca pH 10
• Isoelectric point is around pH 9

Subtilisins - Bacillus origin

• Second largest family of serine proteases
• Alkaline proteases
• Subtilisin Carlsberg - by Bacillus licheniformis
• Widely used in detergents
• Annual production amounts to about 500 tons of pure enzyme protein
• Carlsberg enzyme has a broader substrate specificity než BNP
• Does not depend on Ca2+ for its stability
• Subtilisin Novo
• Bacterial protease Nagase (BPN')
• Produced by Bacillus amyloliquefaciens
• Less commercially important
• Optimal temperature of 60°C
• Optimal pH of 10
• Broad substrate specificity

• serine alkaline protease from the fungus Conidiobolus coronatus
• Distinctly different structure from subtilisin Carlsberg
• Functional similarities [11]

Aspartic proteases - acidic proteases

• Endopeptidases that depend on aspartic acid residues for their catalytic activity
• Three families
• Pepsin (A1)
• Retropepsin (A2)
• Enzymes from pararetroviruses (A3)
• In clan AA
• Maximal activity at low pH (pH 3 to 4)
• Isoelectric points in the range of pH 3 to 4.5
• Members of the pepsin family
• Aspartic proteases
• Inhibited by pepstatin
• Sensitive to diazoketone compounds
• Diazoacetyl-dl-norleucine methyl ester (DAN)
• 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) in the presence of copper ions
• Microbial acid proteases
• Specificity against aromatic or bulky amino acid residues
• Similar to pepsin
• Divided into two groups
• (i) pepsin-like enzymes
• Produced by Aspergillus, Penicillium, Rhizopus, and Neurospora
• (ii) rennin-like enzymes
• Produced by Endothia and Mucor spp. [11]

Cysteine/thiol proteases

• Prokaryotes and eukaryotes
• Cca 20 families of cysteine proteases
• Depends on a catalytic place cysteine and histidine (Cys-His or His-Cys)
• Active only in the presence of reducing agents such as
• HCN
• Cysteine
• Broadly divided into four groups
• (i) papain-like
• (ii) trypsin-like
• (iii) specific to glutamic acid
• (iv) others

• Papain
• Best-known cysteine protease
• Neutral pH optima
• Lysosomal proteases, are maximally active at acidic pH [11]
• Susceptible to sulfhydryl agents such as
• PCMB [11]
• Unaffected by
• DFP
• Metal-chelating agents [11]
• Clostripain
• Anaerobic bacterium Clostridium histolyticum
• Stringent specificity for arginyl residues at the carboxyl side of the splitting bond
• Obligate requirement for calcium
• Isoelectric point of pH 4.9
• Molecular mass of 50 kDa [11]
• Streptopain
• Cysteine protease
• By Streptococcus spp.
• Broader specificity
• Including oxidized insulin B chain
• Other synthetic substrates [11]

Metalloproteases

• Most diverse of the catalytic types of proteases
• Requirement for a divalent metal ion for their activity
• Enzymes from a variety of origins
• Collagenases from higher organisms
• Hemorrhagic toxins from snake venoms
• Thermolysin from bacteria
• About 30 families
• 17 contain only endopeptidases
• 12 contain only exopeptidases
• 1 (M3) contains both endo- and exopeptidases
• Families grouped into different clans
• Amino acid that completes the metal-binding site
• Clan MA - sequence HEXXH-E
• Clan MB - motif HEXXH-H
• Specificity of their action divided into four groups
• Neutral
• Specificity for hydrophobic amino acids
• Alkaline
• Very broad specificity
• Myxobacter I
• Specific for small amino acid residues
• Myxobacter II
• Specific for lysine residue on the amino side
• All of them are inhibited by
• Chelating agents such as
• EDTA
• Not by
• Sulfhydryl agents
• DFP [11]

Thermolysin

• Neutral protease
• Produced by B. stearothermophilus
• Very stable protease
• Half-life of 1 h at 80°C [11]

Collagenase

• Anaerobic bacterium Clostridium hystolyticum
• Aerobic bacterium Achromobacter iophagus
• Other microorganisms including fungi
• Only on collagen and gelatin

Elastase

• By Pseudomonas aeruginosa

Alkaline metalloproteases

• By Pseudomonas aeruginosa
• Serratia spp.
• Active in the pH range from 7 to 9

Myxobacter protease I

• PH optimum of 9.0
• Can lyse cell walls of Arthrobacter crystellopoites
• Protease II cannot lyse the bacterial cells

Matrix metalloproteases

• Prominent role in the degradation of the extracellular matrix during
• Tissue morphogenesis
• Differentiation
• Wound healing
• Cancer
• Arthritis [11]

Nomenclature Committee of the International Union of Biochemistry and Molecular Biology

• Group 3 - hydrolases
• Subgroup 4
• Proteases
• Three major criteria:
• (i) type of reaction catalyzed
• (ii) chemical nature of the catalytic site
• (iii) evolutionary relationship with reference to structure [11]

2 major groups

• Exopeptidases
• Endopeptidases [11]

Exopeptidases

• Cleave the peptide bond proximal to the amino or carboxy termini of the substrate
• Based on their site of action at the N or C terminus
• Aminopeptidases
• Carboxypeptidases [11]

Endopeptidases

• Cleave peptide bonds distant from the termini of the substrate [11]

Functional group - 4 prominent groups

<4>Serine proteases<4>Aspartic proteases<4>Cysteine proteases<4>Metalloproteases

• Few miscellaneous proteases which do not precisely fit into the standard classification
• ATP-dependent proteases which require ATP for activity [11]

Each family of peptidases assigned a code letter denoting the type of catalysis

• S, C, A, M, or U for serine, cysteine, aspartic, metallo-, or unknown type [11]

Examples of extracellular proteases (EPs) include, but are not limited to, matrix metalloproteinases (MMPs), cathepsins, elastase, plasmin, TPA, uPA, kallikrein, ADAMS family members, neprilysin, gingipain, clostripain, thermolysin, serralysin, and other bacterial and viral proteases. While cathepsins are typically present in the lysosome, many of the cathepsins have been shown to play a role in physiological and pathological events occurring extracellularly (Reinheckel T et a Biol Chem 2001;382(5):735-741; Tepel C et a JCeIl ScL 2000 Dec;l 13 Pt 24:4487-98).

Cathepsin B and MMP-9 are extracellular proteases.The term "panel of extracellular proteases," refers to a plurality of distinct extracellular proteases that are used to perform routine assays to monitor the presence or absence of inhibitory activity throughout an extraction process of the invention. A panel typically comprises at least two proteases, but may for some purposes comprise as few as one protease. One skilled in the art would appreciate that as high throughput screening techniques develop, one could routinely assay for the presence or absence of inhibitory activity against as many extracellular proteases as the technology permits.