Inactivation of tyrosine phenol-lyase by Pictet-Spengler reaction and alleviation by T15A mutation on intertwined N-terminal arm.
Citrobacter freundiil-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 degrees C, respectively, which was 15 degrees C lower than when using L-tyrosine as the substrate. The half-lives at 45 degrees 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 (K(m,PLP)) increased slowly when increasing the temperature, while that of the native enzyme showed a sharp increase at temperatures higher than 50 degrees 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.
Lee
Seung-Goo
SG
Systems Microbiology Research Center, KRIBB, Daejeon, Korea.
Hong
Seung-Pyo
SP
Kim
Do Young
DY
Song
Jae Jun
JJ
Ro
Hyeon-Su
HS
Sung
Moon-Hee
MH
D019943:Amino Acid Substitution; D016971:Citrobacter freundii / Q000201:enzymology; D003067:Coenzymes / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007980:Levodopa / Q000378:metabolism* / Q000494:pharmacology; D015394:Molecular Structure; D016296:Mutagenesis; D011485:Protein Binding; D014447:Tyrosine Phenol-Lyase / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism
D003067:Coenzymes; D007980:Levodopa; D014447:Tyrosine Phenol-Lyase