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pre-steady state kinetics and steady state kinetic study, overview. TPL from Citrobacter freundii exhibits two basic pKas, with an average value of 7.8, in the pH dependence of kcat/Km, and kcat is pH-independent. The TPL reaction shows primary isotope effects of about 3 on kcat and 2 on kcat/Km for both enzymes, also suggesting that steps involving transfer of the alpha-proton are partially rate-limiting. The primary isotope effect increases to 5.4 and 3.8, respectively, for kcat and kcat/Km with alpha-[2H]-3-fluoro-L-tyrosine with H343A TPL, compared to 3.9 and 2.2 for wild-type TPL. Stopped-flow measurements, the reaction of TPL with L-tyrosine and 3-fluoro-L-tyrosine in the stopped-flow spectrophotometer also shows rapid formation of quinonoid intermediate |
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