Abstract
The activation energy (EA) and solvent-deuterium kinetic isotope effect (kH/kD) of human skin fibroblast collagenase were studied on the homologous human type I, II, and III collagens in both native and denatured states. Values for EA on human type I and II collagens in solution were 47,000 and 61,000 cal, respectively. The Arrhenius plot for type III collagen, unlike that for the other types, was characterized by a break in EA at approximately 26 degrees C. At temperatures below this point, EA was 42,500 cal; at higher temperatures, EA fell to 29,500 cal. This latter value, intermediate between type I collagen monomers and denatured random gelatin alpha chains, appears to result from a further opening in the already loosened helix of the type III collagen molecule in the region of the 3/4:1/4 collagenase cleavage site. The EA of trypsin on native human type III collagen was also measured and found to be 70,000 cal. This high value calls into question the role of serine proteases in the physiologic degradation of this substrate; a much higher energy expenditure was required for trypsin to cleave type III collagen than for the fibroblast collagenase. Reaction velocity on human collagen types I-III in solution was slowed 15-35% (kH/kD = 1.2-1.5) by the substitution of deuterium for hydrogen in the solvent buffer. This value was far lower than that observed following the aggregation of solution monomers into insoluble fibrils (kH/kD = 9). Denaturation of triple helical monomers into random gelatin alpha chains eliminated any slowing by deuterium, and kH/kD was 1.0 in all cases. Since the same peptide bond hydrolysis accompanies the cleavage of all these forms of the collagen substrate, it would appear that the role of water at the rate-limiting step of collagen degradation may not reside in the hydrolysis of a peptide bond per se, but rather may reflect the difficulty in transporting water molecules to the site of such catalysis, especially following fibril aggregation.
Highlights
Reaction velocity on human collagen types 1-111 in The collagen specificity of humanfibroblast collagenase solution was slowed 15-35% ( k d k D= 1.2-1.5)by the with respect tobothtypeandanimal species of substrate substitution of deuterium for hydrogen in the solvent origin hasalso been examined [3]
There were significant,though more modest, differences attributableto species of substrate origin, the homologous human type I and I11 collagens being more susrather may reflect the difficulty in transporting waterceptible to degradation than theirrespective nonhomologous molecules to the site of such catalysis, especially fol- counterparts
Such studiehsave not been available deuterium kineticisotope effect whichcharacterize the action for other collagen types, especially the homologous human of human skin fibroblast collagenase on guinea pig type I collagens
Summary
Human skinfibroblast procollagenase was purified to homogeneity from serum-containing medium by a combination of ammonium sulfate precipitation, carboxymethylcellulose chromatography, and Ultrogel AcA-44 chromatography, as described by Stricklin et al [5, 6]
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