Cathepsin K, a papain-like human peptidase, is a key enzyme in the process of breakdown of type I collagen – the main bone-forming organic component. Since the disrupted bone homeostasis results in the emergence of numerous diseases such as osteoporosis, certain forms of arthritis and cancer, the inhibition of the enzyme activity is desirable when the enzyme activity is associated with the onset of the disease. Considering that the excessive activity of cathepsin K results in excessive bone breakdown, cathepsin K inhibitors are developed to treat the mentioned diseases. Currently, cathepsin K inhibition is directed towards the binding of inhibitors to the enzyme active site, leading to the complete loss of enzyme activity. Due to the loss of other physiological functions of the active enzyme, the complete inhibition of the activity of cathepsin K may also have undesirable side effects. Thus, it is important to find sufficiently good partial inhibitors of enzyme activity that would inhibit the collagenolytic activity of the enzyme associated with many diseases to a great extent. In the presented thesis, we determined the influence and mechanism of action of selected metal ions (Ce4+, Ga3+,La3+ and Zn2+) on the activity of cathepsin K in three independent tests. First, we determined the influence and mechanism of action of selected metal ions on enzyme activity by monitoring hydrolysis of synthetic substrate Z-FR-AMC. In order to confirm and more precisely define all of the selected inhibition mechanisms, specific velocity diagrams were prepared for all of the selected metal ions, based on which the values of kinetic parameters parameters were determined for each inhibitor. We showed that Ce4+, Ga3+ and La3+ act as hyperbolic or partial inhibitors, more specifically Ce4+ and La3+ by the mechanism of hyperbolic mixed inhibition, and Ga3+ as a hyperbolic competitive inhibitor. Zn2+ ion proved to be linear or complete inhibitors of enzymatic activity, acting by the mechanism of linear competitive inhibition. Since the results obtained with kinetic measurements of degradation of the synthetic substrate do not necessarily apply to natural enzyme substrates, we made a qualitative analysis of the collagenolytic activity of cathepsin K in the presence of high concentrations of selected metal ions. The effects of the inhibitors on the enzymatic activity during the degradation of azocasein were also examined. From the results of the latter experiments, it can be concluded that not all inhibitors act equally well in the presence of different substrates. At the end of this thesis we prepared in situ complexes of biguanide derivatives with Zn2+ ions, because we wanted to check the influence of biguanides on the inhibitory effects of Zn2+ ions. The results of the experiments indicated that the inhibitory effects of Zn2+ ions activity of cathepsin K slightly increased, in complex with phenformin.
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