### Abstract

Tissue inhibitor of metalloproteinase-I (TIMP-1) is a slow, tight- binding inhibitor of fibroblast-type collagenase. Time-course data from inhibition experiments were analyzed by graphic analysis, by nonlinear regression of the analytic integrals of the rate equations and by nonlinear regression with numeric integration of the rate equations. With the same assumptions, approximations and data, all three methods of analysis produced the same model preferences and values for the kinetic parameters. The time- course data for the inhibition of fibroblast-type collagenase by TIMP-1 are best described by the equations for a noncompetitive two-step mechanism, in which an inactive, rapidly formed, reversible complex slowly forms an inactive, tight complex. However, from the analysis of data from experiments at concentrations of TIMP-1 comparable to that of collagenase, it is apparent that free TIMP-1 also functions in the breakdown of the tight complex. The rapidly formed complex has a dissociation constant of 8 nM and reacts to the tight complex with a first-order rate constant of 0.003 s^{-1}. The back reaction of the tight complex to the rapid complex has a second-order rate constant of 5 x 10^{4} M^{-1} s^{-1}. The resulting global dissociation constant of the tight complex is 0.1 nM at 3 nM TIMP-1 and collagenase concentration. Collagenase without the carboxyl-terminal domain (mini-collagenase) is inhibited by TIMP-1 according to a mechanism, in which the rapidly formed complex has such a high dissociation constant (247 nM) that it effectively constitutes a one-step mechanism, in which TIMP-1 binds with an apparent second-order rate constant of 9.6 x 10^{4} mol^{-1} s^{-1} and the enzyme-TIMP-1 complex dissociates with a first order rate constant of 0.00026 s^{-1}. The apparent global dissociation constant for the tight complex (2.7 nM) is higher than that for the fibroblast-type collagenase. Participation of TIMP- 1 in the dissociation is not demonstrable. Therefore, the carboxyl-terminal domain of fibroblast-type collagenase is important for the initial, rapid binding of TIMP-1 and the initial complex contributes to the overall binding.

Original language | English (US) |
---|---|

Pages (from-to) | 23938-23945 |

Number of pages | 8 |

Journal | Journal of Biological Chemistry |

Volume | 271 |

Issue number | 39 |

DOIs | |

State | Published - 1996 |

Externally published | Yes |

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### ASJC Scopus subject areas

- Biochemistry

### Cite this

*Journal of Biological Chemistry*,

*271*(39), 23938-23945. https://doi.org/10.1074/jbc.271.39.23938

**The mechanism of inhibition of collagenase by TIMP-1.** / Taylor, Kenneth B.; Windsor, L.; Caterina, Nancy C M; Kirby Bodden, M.; Engler, Jeffrey A.

Research output: Contribution to journal › Article

*Journal of Biological Chemistry*, vol. 271, no. 39, pp. 23938-23945. https://doi.org/10.1074/jbc.271.39.23938

}

TY - JOUR

T1 - The mechanism of inhibition of collagenase by TIMP-1

AU - Taylor, Kenneth B.

AU - Windsor, L.

AU - Caterina, Nancy C M

AU - Kirby Bodden, M.

AU - Engler, Jeffrey A.

PY - 1996

Y1 - 1996

N2 - Tissue inhibitor of metalloproteinase-I (TIMP-1) is a slow, tight- binding inhibitor of fibroblast-type collagenase. Time-course data from inhibition experiments were analyzed by graphic analysis, by nonlinear regression of the analytic integrals of the rate equations and by nonlinear regression with numeric integration of the rate equations. With the same assumptions, approximations and data, all three methods of analysis produced the same model preferences and values for the kinetic parameters. The time- course data for the inhibition of fibroblast-type collagenase by TIMP-1 are best described by the equations for a noncompetitive two-step mechanism, in which an inactive, rapidly formed, reversible complex slowly forms an inactive, tight complex. However, from the analysis of data from experiments at concentrations of TIMP-1 comparable to that of collagenase, it is apparent that free TIMP-1 also functions in the breakdown of the tight complex. The rapidly formed complex has a dissociation constant of 8 nM and reacts to the tight complex with a first-order rate constant of 0.003 s-1. The back reaction of the tight complex to the rapid complex has a second-order rate constant of 5 x 104 M-1 s-1. The resulting global dissociation constant of the tight complex is 0.1 nM at 3 nM TIMP-1 and collagenase concentration. Collagenase without the carboxyl-terminal domain (mini-collagenase) is inhibited by TIMP-1 according to a mechanism, in which the rapidly formed complex has such a high dissociation constant (247 nM) that it effectively constitutes a one-step mechanism, in which TIMP-1 binds with an apparent second-order rate constant of 9.6 x 104 mol-1 s-1 and the enzyme-TIMP-1 complex dissociates with a first order rate constant of 0.00026 s-1. The apparent global dissociation constant for the tight complex (2.7 nM) is higher than that for the fibroblast-type collagenase. Participation of TIMP- 1 in the dissociation is not demonstrable. Therefore, the carboxyl-terminal domain of fibroblast-type collagenase is important for the initial, rapid binding of TIMP-1 and the initial complex contributes to the overall binding.

AB - Tissue inhibitor of metalloproteinase-I (TIMP-1) is a slow, tight- binding inhibitor of fibroblast-type collagenase. Time-course data from inhibition experiments were analyzed by graphic analysis, by nonlinear regression of the analytic integrals of the rate equations and by nonlinear regression with numeric integration of the rate equations. With the same assumptions, approximations and data, all three methods of analysis produced the same model preferences and values for the kinetic parameters. The time- course data for the inhibition of fibroblast-type collagenase by TIMP-1 are best described by the equations for a noncompetitive two-step mechanism, in which an inactive, rapidly formed, reversible complex slowly forms an inactive, tight complex. However, from the analysis of data from experiments at concentrations of TIMP-1 comparable to that of collagenase, it is apparent that free TIMP-1 also functions in the breakdown of the tight complex. The rapidly formed complex has a dissociation constant of 8 nM and reacts to the tight complex with a first-order rate constant of 0.003 s-1. The back reaction of the tight complex to the rapid complex has a second-order rate constant of 5 x 104 M-1 s-1. The resulting global dissociation constant of the tight complex is 0.1 nM at 3 nM TIMP-1 and collagenase concentration. Collagenase without the carboxyl-terminal domain (mini-collagenase) is inhibited by TIMP-1 according to a mechanism, in which the rapidly formed complex has such a high dissociation constant (247 nM) that it effectively constitutes a one-step mechanism, in which TIMP-1 binds with an apparent second-order rate constant of 9.6 x 104 mol-1 s-1 and the enzyme-TIMP-1 complex dissociates with a first order rate constant of 0.00026 s-1. The apparent global dissociation constant for the tight complex (2.7 nM) is higher than that for the fibroblast-type collagenase. Participation of TIMP- 1 in the dissociation is not demonstrable. Therefore, the carboxyl-terminal domain of fibroblast-type collagenase is important for the initial, rapid binding of TIMP-1 and the initial complex contributes to the overall binding.

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UR - http://www.scopus.com/inward/citedby.url?scp=0029796959&partnerID=8YFLogxK

U2 - 10.1074/jbc.271.39.23938

DO - 10.1074/jbc.271.39.23938

M3 - Article

VL - 271

SP - 23938

EP - 23945

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 39

ER -