Borivoj Keil

Purification stability and inhibition of the collagenase from Achromobacter iophagus

An aerobic microorganism, Achromobacter iophagus, which was isolated from cured hides and which is a source collagenolytic activity has bee11 described in a series of papers [l-3]. The crude collagenase from Achromobacter is now available on a larger scale; this gave us an opportunity to study its further purification, as well as its enzymatic, pharmacological and immunogenic properties. In this paper purification is described leading to a collagenase of specific activity of 1587 nkat/mg, the highest activity obtained as yet for any collagenase. p-Chloromercuribenzoate removes most of the caseinolytic activity in the crude enzyme. EDTA, cysteine and histidine inhibit the enzyme, Dip-F, N-ethylmaleimide and ClHgBzOH are without effect. This localizes the enzyme as a metalloenzyme (EC 3.4.24).

Hypodermin A, a trypsin-like neutral proteinase from the insect Hypoderma lineatum.

Hypodermin A, a serine proteinase from the larva Hypoderma lineatum, with a molecular weight of 27 000 was obtained in pure form by ion-exchange chromatography. It is inhibited by diisopropyl phosphofluorate, a serine proteinase inhibitor, but not by metallo or cysteine enzyme inhibitors such as EDTA or thiol reagents. In the same way, it is fully inactivated by trypsin inhibitors, but not by specific chymotrypsin inhibitors. Its specificity, limited to carboxyl side of arginine residue in B-chain of insulin, is more complicated on other polypeptide substrates. Sequence analysis suggests structural homology with H. lineatum collagenase as well as with other members of the trypsin family.

Some newly characterized collagenases from procaryotes and lower eucaryotes.

SummaryChemical and enzymatic properties of four collagenases newly isolated from anaerobic Clostridium histolyticum, aerobic Achromobacter iophagus, and from two lower eucaryotes, the fungus Entomophthora coronata and the insect Hypoderma lineatum are reviewed.The problems of their biosynthesis and precursors, namely the effect of induction of collagenase and neutral proteinase in Achromobacter by their macromolecular substrates are discussed.The two bacterial collagenases are Zn-metallo-enzymes; the highly purified Clostridium collagenase contains cyst(e)ine, serine phosphate and tryptophan additionally to amino acids reported previously. Achromobacter collagenase has the highest specific activity of all collagenases; it yields by autolysis enzymatically active degraded forms. The active dimer is composed of two identical subunits of molecular weight 35,000. Similarities between Achromobacter collagenase, thermolysin and Bacillus subtilis neutral proteinase in molecular weight, amino acid composition, and amino acids important for the active sites are discussed.The two collagenases from low eucaryotes are serine proteinases; Hypoderma collagenase is homologous to the trypsin family in the amino terminal sequence.The initial cleavage of native collagen by highly purified bacterial collagenases occurs in the central helical part of the a chains and not progressively from the amino terminal end. One of the two initial cleavages produced by Achromobacter collagenase is situated in the region cleaved specifically by vertebrate collagenases, but with different bond specificity. The same is true for the insect collagenase. Entomophthora collagenase is a proteinase of broad specificity which also cleaves collagen in its helical parts. All four collagenases also degrade other proteins according to their bond specificity.

Specificity of the collagenolytic enzyme from the fungus Entomophthora coronata: Comparison with the bacterial collagenase from Achromobacter iophagus☆

Abstract The specificity of the collagenolytic enzyme from the fungus Entomophthora coronata toward some inhibitors and the B chain of oxidized insulin was investigated and compared to that of the bacterial collagenase from Achromobacter iophagus. The fungal enzyme was completely inhibited by diisopropylfluorophosphate, tosyl- l -lysine chloromethyl ketone, and tosyl-amino-2-phenylethyl chloromethyl ketone but not at all by ethylenediaminetetraacetate. This indicates that it is not a metalloenzyme like the bacterial Achromobacter collagenase. The B chain of insulin was not hydrolysed at all by the bacterial enzyme under conditions where extensive digestion was observed with the Entomophthora enzyme. The fungal enzyme cleaves preferentially the bonds Leu 15 - Tyr 16 and Leu 11 Val 12 as determined by automatic sequencing; the secondary cleavages were identified by a systematic analysis of the digestion mixture; thus, the fungal collagenolytic enzyme from Entomophthora coronata differs both structurally and functionally from the bacterial Achromobacter collagenase.

Specificity of collagenase from Achromobacter iophagus

Many enzymes which have been originally described as collagenases, were shown later to be either proteases or peptidases of broad or different specificity. A true collagenase degrades in the helical regions of the native collagen preferentially the bond Y-Gly. The observations that an enzyme degrades synthetic peptide of a composition similar to sequences existing in collagen or that it degrades collagen are not sufficient evidence that it is a collagenase. A new collagenolytic enzyme, synthesized by a nonpathogenic, aerobic strain of Achromobacter iophagus has been first described by Woods and Welton [ 1,2] . Recently this enzyme has been obtained in the homogeneous state on a preparative scale in our laboratory [3,4] . Its enzymatic activity using both collagen and synthetic substrates is many times higher than the purest samples of the collagenases from Clostridium histolyticum. Both theoretical and practical interest in this highly active, non-toxic enzyme prompted us to examine its specificity, namely in comparison with the widely studied Clostridium collagenase. According to the generally accepted scheme, the Clostridium enzyme cleaves in the helical regions of native collagen predominantly the bond Y-Gly in sequences of the type -Pro-Y-GlyPro, where Y is most frequently a neutral amino acid [5,6]. The enzyme cleaves readily this kind of bond also in many synthetic peptidic substrates [7,X]. The interpretation of the results is often handicapped by the complex nature of the collagenase’ samples used for the studies: chromatographically purified commercial collagenases are heterogeneous, and further purifications undertaken by different authors yielded varying num-

The induction of collagenase and a neutral proteinase by their high molecular weight substrates in Achromobacter iophagus

The synthesis of collagenase in Achromobacter isophagus has been shown to be inducible by denatured collagen and by its high molecular weight fragments. The presence in the macromolecular inducer of peptide bonds which could be digested by the collagenase is indispensable for the effect of induction. On the other hand, an addition of a low molecular weight substrate or inhibitor of collagenase does not stimulate the enzyme synthesis. Lack of collagenase induction was also observed in the case of β-casein which is a macromolecular substrate with four peptide bonds digestible by the collagenase. Nevertheless in the presence of β-casein the induction of a neutral caseinolytic proteinase was found. The probable role of conformation structure of a macromolecular substrate in the mechanism of induction is discussed. The dependence of induction on the growth phase of the culture was studied. The collagenase activity appears only after the last phase of the exponential growth. It was proved that no zymogen or cell-accumulated enzyme is present in the first stage of exponential growth and that the collagenase synthesis is in direct correlation with a particular state of the bacterial growth cycle.

Subunit structure of Arhromobacter collagenase

Abstract The highly active form of collagenase (EC 3.4.24.3) from Achromobacter iophagus (specific activity 2 μkat/mg) has a molecular weight of 70 000 and the sedimentation coefficient s20,w = 4.4 S. It is composed of two subunits of molecular weight 35 000 and s20,w of 2.9 S. The dissociation of the dimer under different conditions resulted in the complete and irreversible loss of enzymic activity. A unique N-terminal sequence Thr-Ala-Ala-Asp-Leu-Glu-Ala-Leu-Val- indicates that the two subunits are identical, at least in the N-terminal part of the polypeptide chain. Reduction and pyridylethylation of the subunit change neither molecular weight nor amino acid composition: therefore each subunit of molecular weight 35 000 consists of a single polypeptide chain. Another active and homogeneous form of Achromobacter collagenase (specific activity 1.64 μkat/mg) gives a value for the apparent molecular weight of 80 000 on sodium dodecyl sulphate-polyacrylamide electrophoresis. It is also a dimer in which each of the two subunits of molecular weight 35 000 binds non-covalently a peptide of molecular weight 5000. The dissociation of this form of collagenase is also accompanied by irreversible loss of enzymic activity. The amino acid composition of the subunits which were isolated from both 70 000 and 80 000 collagenases is the same. The role of dimer-monomer equilibrium in the biological function of collagenase is discussed.

Five sepharose‐bound ligands for the chromatographic purification of Clostridium collagenase and clostripain

Two of the extracellular proteolytic enzymes produced by the anaerobic microorganism Clostridium histolyticum have been studied in particular because of their restricted specificity: collagenase (clostridiopeptidase A, EC 3.4.24.3) which cleaves native collagen preferentially at the amino group of glycine residues and clostripain (clostridiopeptidase B, EC 3.4.22.8) which cleaves proteins exclusively at the carboxyl of arginine residues. Several methods have been proposed for their separation and purification: the highest specific esterolytic activity values as yet obtained for clostripain were 1 .l. pkat.mg-’ [l] and 2.72 pkat.mg-’ (after activation) [2] ; the highest value of specific activity of purified collagenase on a synthetic substrate [3] was 0.27 pkat.mg-’ [4]. In both cases these values can still not be considered as upper limits. In this paper we describe several chromatographic separations using Sepharose-bound ligands: compounds which act as competitive inhibitors of clostripain, such as arginine [5], polyarginine [l] , benzamidine [6] and soybean trypsin inhibitor [7], the SH-binding mercurial p-aminophenylmercuric acetate [8] and acridme dye Rivanol which is known to inhibit collagenase [9] .

Evidence for an active-center cysteine in the SH-proteinase α-clostripain through use of N-tosyl-L-lysine chloromethyl ketone

The rapid reaction of α‐clostripain with tosyl‐L‐lysine chloromethyl ketone results in a complete loss of activity and in the disappearance of one titratable SH group whereas the number of histidine residues is not affected. Tosyl‐L‐phenylalanine chloromethyl ketone and phenylmethylsulfonyl fluoride have no effect on the catalytic activity. From the molar ratio and under the assumption of 1:1 molar interaction, the fully active enzyme has a specific activity of 650–700 [twice the value proposed by Porter et al. (J. Biol. Chem. 246 (1971) 7675‐7682)]. Partial oxidation makes it experimentally impossible to attain this maximal value.

Circular dichroism comparative studies of two bacterial collagenases and thermolysin

The recently isolated and purified collagenase produced by Achromobacter iophagus, the collagenase from Clostridium histolyticum, and thermolysin, three enzymes having common properties, were studied by circular dichroism. From the spectra of the aqueous solutions obtained in the peptide region, the fraction of alpha helix, beta sheet and aperiodic segments in the three proteins could be estimated. Good similarity was found between Achromobacter collagenase and thermolysin, which both contain a high fraction of alpha helix. Side-chain contributions were analyzed in the aromatic region of thespectra: effects of pH and of organic solvents were observed, showing the strong influence of surroundings on the stabilization of the proteins.