Inactivation of tyrosine phenol-lyase by Pictet-Spengler reaction and alleviation by T15A mutation on intertwined N-terminal arm = Pictet-Spengler 반응 Tyrosine Phenol-Lyase의 불활성화 및 N-말단 Arm

Abstract

Citrobacter freundii l-tyrosine phenol-lyase (TPL) was inactivated by a Pictet-Spengler reaction between the cofactor and a substrate, 3,4-dihydroxyphenyl-l-alanine (l-dopa), in proportion to an increase in the reaction temperature. Random mutagenesis of the tpl gene resulted in the generation of a Thr15 to Ala mutant (T15A), which exhibited a two-fold improved activity towards l-DOPA as the substrate. The Thr15 residue was located on the intertwined N-terminal arm of the TPL structure, and comprised an H-bond network in proximity to the hydrophobic core between the catalytic dimers. The maximum activity of the mutant and native enzymes with l-DOPA was detected at 45 and 40°C, respectively, which was 15°C lower than when using l-tyrosine as the substrate. The half-lives at 45°C were about 16.8 and 6.4 min for the mutant and native enzymes, respectively, in 10 mm l-DOPA. On treatment with excess pyridoxal-5′-phosphate (PLP), the l-DOPA-inactivated enzymes recovered over 80% of their original activities, thereby attributing the inactivation to a loss of the cofactor through Pictet-Spengler condensation with l-DOPA. Consistent with the extended half-life, the apparent Michaelis constant of the T15A enzyme for PLP (Km,PLP) increased slowly when increasing the temperature, while that of the native enzyme showed a sharp increase at temperatures higher than 50°C, implying that the loss of the cofactor with the Pictet-Spengler reaction was prevented by the tighter binding and smaller release of the cofactor in the mutant enzyme.