Elementary Processes in the Interaction of Tyrosine Phenol Lyase with Inhibitors and Substrate

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Abstract
The binding process of tyrosine phenol-lyase, a B6,-enzyme, with inhibitors (L-alanine, D-alanine, and L-meta-tyrosine) or the substrate (L-tyrosine) was investigated kinetically by the stopped-flow method. Since a new absorption spectrum at around 500 nm appears upon binding of the enzyme with the inhibitors or the substrate, the reactions were monitored by means of the visible spectral change. For the enzyme-inhibitor interaction, the concentration dependence of the reciprocal relaxation time observed by the stopped-flow method was analyzed and indicated a two-step mechanism, a fast bimolecular association followed by a slow uni-molecular process, where EI1 and EI2 are structural isomers of the enzyme-inhibitor complexes and k1k−1.etc., are rate constants. No visible spectral change was detected by the rapid-scan stopped-flow method in the fast association process between the enzyme and the inhibitors. In the case of L-alanine and L-meta-tyrosine, an additional very slow relaxational process was observed, preceded by the fast process. The reciprocal relaxation time was independent of the inhibitor concentration. Upon binding of the enzyme with substrate, L-tyrosine, three separate relaxational processes with visible spectral changes were observed. By analysis of the concentration dependence of the reciprocal relaxation time of the fastest process, the association and dissociation rate constants were determined (K1=1.4×104M−1·S−1 and K−1=7.8S−1) The concentration dependence of the reciprocal relaxation time for the middle process showed a saturation curve and the rate constants K2(=0.14S−1) and k−1=0.15S−1) were determined assuming the above two-step mechanism. The value of k2 was nearly equal to the catalytic center activity, kcat (=0.50 S−1), suggesting that the rate-determining step could be the first unimolecular process. The reciprocal relaxation time for the slowest relaxation process did not depend on the substrate concentration. The slowest process was assigned to the formation of an abortive intermediate branching from the main catalytic pathway.

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