Fig. 2.

Equilibration of enzyme–substrate conformational transitions before steady state. (A) Transient kinetics fluorescence analyses showing the fluorescence emission after mixing MT1-MMP and FRET triple-helical (blue) and single-stranded (red) substrates in a stopped-flow apparatus (λ_excitation = 340 nm; λ_emission > 380 nm). (B) Time-dependent changes in intrinsic Trp and Tyr fluorescence of MT1-MMP measured after mixing MT1-MMP with triple-helical (blue) and single-stranded (red) substrate with λ_excitation = 295 nm and λ_emission = 320 nm. The experiments were conducted under identical conditions to A. (C and D) XAS analysis shows differences in interactions with the zinc ion and the two different model substrates. The X-ray absorption data are presented in the form of Fourier transform (FT) spectra to provide the radial distribution of the atoms within the first and second coordination shells of the catalytic zinc ion in MT1-MMP during hydrolysis of (C) triple-helical or (D) single-stranded substrates. The shape and amplitude of the Fourier transform peaks are directly related to the type and number of amino acid residues in the immediate vicinity of the zinc ion. The increase in the first shell amplitude is correlated with an increase in coordination number. (C) A stable pentacoordinated zinc peptide intermediate is rapidly formed during hydrolysis of the triple-helical substrate. (D) On the contrary, transient binding of the single-stranded substrate is detected before the formation of the stable Michaelis complex.