Rapid determination of substrate specificity of Clostridium histolyticum beta-collagenase using an immobilized peptide library.

The molecular basis of the substrate specificity of Clostridium histolyticum beta-collagenase was investigated using a combinatorial method. An immobilized positional peptide library, which contains 24,000 sequences, was constructed with a 7-hydroxycoumarin-4-propanoyl (Cop) fluorescent group attached at the N terminus of each sequence. This immobilized peptide library was incubated with C. histolyticum beta-collagenase, releasing fluorogenic fragments in the solution phase. The relative substrate specificity (k(cat)/K(m)) for each member of the library was determined by measuring fluorescence intensity in the solution phase. Edman sequencing was used to assign structure to subsites of active substrate mixtures. Collectively, the substrate preference for subsites (P(3)-P(4)') of C. histolyticum beta-collagenase was determined. The last position on the C-terminal side in which the identity of the amino acids affects the activity of the enzyme is P(4)', and an aromatic side chain is preferred in this position. The optimal P(1)'-P(3)' extended substrate sequence is P(1)'-Gly/Ala, P(2)'-Pro/Xaa, and P(3)'-Lys/Arg/Pro/Thr/Ser. The Cop group in either the P(2) or P(3) position is required for a high substrate activity with C. histolyticum beta-collagenase. S(2) and S(3) sites of the protease play a dominant role in fixing the substrate specificity. The immobilized peptide library proved to be a powerful approach for assessing the substrate specificity of C. histolyticum beta-collagenase, so it may be applied to the study of other proteases of interest.     

A novel aminopeptidase from Clostridium histolyticum

An aminopeptidase was found in the culture filtrate of Cl. histolyticum and purified to homogeneity (130 times) in a two-step procedure. All types of N-terminal amino acids, including proline and hydroxyproline are cleaved by the enzyme from small peptides and from polypeptides. A low rate of hydrolysis was observed for β-naphthylamides and for alanine amide; p-nitroanilides were not hydrolyzed. Kinetic parameters (K_m and V_max) for several tripeptides and the tetrapeptide Pro-Gly-Pro-Pro were determined. The enzyme has a pH optimum at 8.6. The presence of either Mn⁺⁺ or Co⁺⁺ is essential for its activity. Only slight activation was observed with Ni⁺⁺ and Cd⁺⁺, while Zn⁺⁺ and Cu⁺⁺ were inhibitory. The molecular weight of the native enzyme is about 340,000, and a molecular weight of about 60,000 was determined for the reduced and denatured enzyme by gel electrophoresis in sodium dodecyl sulfate (SDS).The culture filtrate of Cl. histolyticum has been shown to contain various proteolytic enzymes, in addition to collagenase^1–5. In a search for enzymes acting on proline-rich peptides, we tested the crude filtrate with (Pro-Gly-Pro)_n, (Pro-Gly-Pro)_n-OMe, α,DNP-(Pro-Gly-Pro)_n and poly-L-proline as substrates. Proline was formed only from (Pro-Gly-Pro)_n and its methyl ester. This showed the presence in Cl. histolyticum filtrate of an aminopeptidase which cleaves N-terminal proline from polypeptides but not from polyproline. The purification and some of the properties of this clostridial aminopeptidase (CAP) are described in this communication.     

Substrate recognition by the collagen-binding domain of Clostridium histolyticum class I collagenase

Clostridium histolyticum type I collagenase (ColG) has a segmental structure, S1+S2+S3a+S3b. S3a and S3b bound to insoluble collagen, but S2 did not, thus indicating that S3 forms a collagen-binding domain (CBD). Because S3a+S3b showed the most efficient binding to substrate, cooperative binding by both domains was suggested for the enzyme. Monomeric (S3b) and tandem (S3a+S3b) CBDs bound to atelocollagen, which contains only the collagenous region. However, they did not bind to telopeptides immobilized on Sepharose beads. These results suggested that the binding site(s) for the CBD is(are) present in the collagenous region. The CBD bound to immobilized collagenous peptides, (Pro-Hyp-Gly)(n) and (Pro-Pro-Gly)(n), only when n is large enough to allow the peptides to have a triple-helical conformation. They did not bind to various peptides with similar amino acid sequences or to gelatin, which lacks a triple-helical conformation. The CBD did not bind to immobilized Glc-Gal disaccharide, which is attached to the side chains of hydroxylysine residues in the collagenous region. These observations suggested that the CBD specifically recognizes the triple-helical conformation made by three polypeptide chains in the collagenous region.     

Toxigenicity of Clostridium histolyticum

Nishida, Shoki (Kanazawa University, Kanazawa, Japan), and Masaaki Imaizumi. Toxigenicity of Clostridium histolyticum. J. Bacteriol. 91:477-483. 1966.-From 234 soil samples, 21 strains of Clostridium histolyticum of different levels of alpha-toxigenicity were isolated by a new method specially designed for the isolation of this species. The alpha-toxigenicity of freshly isolated strains and of stock strains was closely associated with the potentiality for sporulation, growth, and smooth-colony formation. The presence of sugars, particularly xylose and arabinose, was inhibitory for growth. A few controversies on the biological properties of this species seem to be due to disregard for the growth-inhibiting effects of these sugars.     

An extracellular aminopeptidase from Clostridium histolyticum.

An aminopeptidase was isolated from the culture filtrate of Clostridium histolyticum and purified to homogeneity. Absence of endopeptidase activity in the purified preparation was demonstrated. Gel filtration on a calibrated column indicates an apparent molecular weight of 340000 for the native enzyme. Gel electrophoresis of the denatured enzyme in the presence of dodecylsulfate in constant acrylamide concentration and in a concentration gradient, resulted in the appearance of a single component for which a molecular weight of 51000 and 59000 respectively, was calculated. From mobilities of crosslinked and denatured protein species a molecular weight of 56000 was obtained for the monomer. Specificity studies show that the enzyme cleaves all types of N-terminel amino acid residues including proline and hydroxyproline from small peptides and from polypeptides. The peptide bond formed between an N-terminal amino acid residue and proline is not cleaved by the enzyme. The combined action of aminopeptidase-P and clostridal aminopeptidase leads to complete hydrolysis of the proline-rich nonapeptide bradykinin. Low rates of hydrolysis was observed for charged residues, and amides of amino acids. Kinetic studies with five tripeptides of the general structure X-Gly-Gly, where X stands for Leu, Phe, Val, Ala, or Pro, show a decrease in Km with the increasing size of the hydrophobic side chain of X. The highest Kcat values are observed with proline and alanine. In the series Pro-Gly, Pro-Gly-Pro, Pro-Gly-Pro-Pro, the last peptide is the best substrate, indicating an active site complementary to at least four amino acid residues. The enzymatic activity is dependent on the presence of divalent cations, maximal activation being reached with Mn2+ and Co2+. The optimal pH for the Mn2+ and Co2+- activated enzyme is 8.6 and 8.2 respectively. The optimal temperature is 40 degrees C. Inhibition of the aminopeptidase was achieved with Zn2+, Cu2+ and p-mercuribenzoate, but not with diisopropylphosphofluoridate.     

Characterization of the individual collagenases from Clostridium histolyticum

The six collagenases (alpha, beta, gamma, delta, epsilon, and zeta) from Clostridium histolyticum isolated in the preceding paper [Bond, M. D., & Van Wart, H. E. (1984) Biochemistry (first paper of three in this issue)] have been characterized in detail. The molecular weights determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis range from 68 000 to 125 000. Isoelectric focusing experiments demonstrate that the isoelectric points of the collagenases are in the 5.35-6.20 range. These experiments also reveal that the subspecies of alpha- and gamma-collagenases (alpha1 vs. alpha 2 and gamma 1 vs. gamma 2) have different isoelectric points but the same molecular weights. Microheterogeneity is also observed for the beta- and epsilon-collagenases. The amino acid compositions of all six collagenases have been determined, and analysis for neutral sugars and hexosamines shows that none of the enzymes have a significant carbohydrate content. Zinc and calcium are the only metals that copurify with the collagenases. The purified enzymes contain approximately 1 mol of zinc/mol of protein and a calcium content that varies from about 2 mol/mol for alpha-collagenase to about 7 mol/mol for beta-collagenase. All of the collagenases are 5-10 times more active against gelatin than collagen. The alpha-, beta-, and gamma-collagenases are significantly less active toward the synthetic peptide substrates examined than the delta-, epsilon, and zeta-collagenases. This property, taken together with data on the stabilities and amino acid compositions of these enzymes, strongly supports their assignment to two distinct classes. This establishes clearly that C. histolyticum does, indeed, produce more than one different type of collagenase.     

Complement-inactivating Proteinase(s) from Clostridium histolyticum

A proteinase fraction inhibiting the hemolytic activity of guinea pig complement was obtained from supernatant fluids of Clostridium histolyticum cultures and purified 150- to 350-fold by ammonium sulfate precipitation, Sephadex G-75 gel filtration, and diethylaminoethyl cellulose chromatography. An assay was developed based on the inactivation of hemolytic complement. Partially purified anticomplementary preparations were active against casein and were capable of "solubilizing" Escherichia coli endotoxin. Two components were found by differential heat inactivation, with complement and casein as substrates, but only one of these components was active against endotoxin. The more heat-stable activity, showing 50% inactivation at about 47 C, was characterized as to pH and ionic strength optima and sensitivity to reagents such as cysteine, beta-mercaptoethanol, ethylenediaminetetraacetate, and heavy metals.     

Glycine fermentation by Clostridium histolyticum

Clostridium histolyticum grew on glycine, arginine, or threonine as sole substrate. Arginine degradation preceded that of glycine and partially inhibited that of threonine when two amino acids were present. Each amino acid seemed to be individually catabolized, not by a Stickland type of reaction. Glycine fermentation required the presence of complex ingredients. Therefore, an effect of selenite on glycine catabolism could only be demonstrated after scavenging selenium contamination by preculturing Peptostreptococcus glycinophilus in that medium. C. acidiurici was not suited as selenium accumulating organism as C. histolyticum was inhibited by the residual uric acid. Arginine catabolism was unaffected by seleniuum depriviation. The labelling pattern obtained in acetate after incubation of C. histolyticum with [1-¹⁴C]- or [2-¹⁴C]glycine strongly indicated the metabolism of glycine via the glycine reductase pathway.     

Substrate competition and specificity at the active site of amylopullulanase from Clostridium thermohydrosulfuricum

A highly thermostable pullulanase purified from Clostridium thermohydrosulfuricum strain 39E displayed dual activity with respect to glycosidic bond cleavage. The enzyme cleaved alpha-1,6 bonds in pullulan, while it showed alpha-1,4 activity against malto-oligosaccharides. Kinetic analysis of the purified enzyme in a system which contained both pullulan and amylose as the two competing substrates were used to distinguish the dual specificity of the enzyme from the single substrate specificity known for pullulanases and alpha-amylases.