Characteristics of the action of human skin fibroblast collagenase on fibrillar collagen.

The characteristics of the binding of pure human skin fibroblast collagenase to the in vivo form of its substrate, fibrillar collagen, has been studied under in vitro conditions. Collagenase binds tightly to reconstituted native collagen fibrils and appears to remain bound to the substrate throughout ongoing collagen degradation at 37"C, independent of subsequent dilution with buffer or the addition of exogenous collagen as a competitor. Therefore, during the degradation of fibrillar collagen, no equilibrium appears to exist between collagenase molecules bound to fibrillar substrate and the buffer of the reaction mixture. The binding of collagenase to fibrillar collagen at 25°C or 37°C displays saturation kinetics. At 25"C, binding is complete within 20 min, during which time collagen degradation is virtually absent. By analyzing the amount of bound enzyme as a function of collagenase concentration, the apparent Kd was determined to be 9.5 X lo" M. These same studies revealed that only 10% of the collagen molecules in the fibrillar substrate gel appear to be accessible to enzyme for binding, prior to degradation of the substrate. Despite the presence of a solid Substrate, additional binding data indicate that, under appropriate volume conditions, equations derived from classical solution kinetics can be used as a model to rather accurately predict enzyme binding, provided that the number of substrate molecules available for binding is defined as equal to 10% of the total number of collagen molecules. It would, therefore, appear that collagen fibrils are composed of two sets of molecules: those that are initially available for collagenase binding, located on or near the surface of each individual fibril, and which comprise approximately 10% of the total, while the remaining 90% of molecules must become accessible to enzyme only during catalytic degradation of the substrate. Finally, the turnover number of human skin fibroblast collagenase, with guinea pig type I fibrillar collagen as substrate, has been determined. Approximately 25 molecules of collagen are degraded per molecule of collagenase per h.