Human skin fibroblast collagenase: interaction with substrate and inhibitor.
Human skin fibroblasts secrete collagen, procollagenase and a collagenase inhibitor. This study addresses the nature of the interaction between these important fibroblast products. The binding of procollagenase and of active collagenase to native collagen in solution was examined by employing Sephadex G-150 gel-filtration chromatography to separate bound versus unbound enzyme. Active enzyme bound readily to collagen; the equilibrium constant of binding, Kd, was calculated to be 5.1 to 10(-7)M. Thus, collagenase binds with nearly equal affinity to both monomeric collagen and aggregated fibrils (Kd = 9 X 10(-7)M; [Welgus et al., 1980]). Furthermore, since Kd congruent to Km congruent to 10(-6)M, the ratio k2/k1 must be extremely small, directly implicating the catalytic step represented by the rate constant k2, and not the binding of enzyme to substrate, as the rate-limiting step of collagenase action. In contrast, procollagenase demonstrated no capacity to bind to collagen. The interaction of procollagenase and of active collagenase with inhibitor was examined utilizing both conventional and high-precision liquid gel-filtration chromatography. A higher molecular weight complex could be demonstrated consisting of active collagenase and inhibitor; no such interaction occurred between procollagenase and the inhibitory protein. Analysis of Lineweaver-Burk plots showed that inhibition was accompanied by a corresponding change in Vmax; Km remained unchanged. Such results are indicative of a noncompetitive mechanism of inhibition and are consistent with the formation of an enzyme-inhibitor complex. The Ki of enzyme-inhibitor binding was determined to be less than 10(-9)M. The data indicate that procollagenase can neither interact with its specific inhibitor nor bind to collagen. Extracellular activation of the collagenase zymogen is thus a critical event, which can be followed either by binding to substrate or interaction with inhibitor.
Welgus
H G
HG
Jeffrey
J J
JJ
Eisen
A Z
AZ
Roswit
W T
WT
Stricklin
G P
GP
D002850:Chromatography, Gel; D003094:Collagen / Q000378:metabolism*; D017364:Collagenases; D004791:Enzyme Inhibitors / Q000378:metabolism*; D004792:Enzyme Precursors / Q000378:metabolism*; D005347:Fibroblasts / Q000201:enzymology* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D003012:Microbial Collagenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008970:Molecular Weight; D012867:Skin / Q000378:metabolism; D019714:Tissue Inhibitor of Metalloproteinases
D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D019714:Tissue Inhibitor of Metalloproteinases; D003094:Collagen; D017364:Collagenases; C022440:procollagenase; D003012:Microbial Collagenase