L-Tyrosine, 2-chloro-L-tyrosine, 2-bromo-L-tyrosine, and 2-iodo-L-tyrosine were synthesized by β-tyrosinase obtained from cells of Escherichia intermedia A-21, through the reversal of the α,β-elimination reaction, and their molecular structures were analyzed by element analysis, NMR spectroscopy, mass spectrometry and IR spectroscopy. Rates of synthesis and hydrolysis of halogenated tyrosines by β-tyrosinase, inhibition of the enzyme activity by halogenated phenols, and effects of addition of m-bromophenol on the synthesis of 2-bromotyrosine were determined. The results obtained were as follows:
1) In the synthesis of halogenated tyrosines, the yield of 2-chlorotyrosine from m-chlorophenol were approximately 15 per cent, that of 2-bromotyrosine from m-bromophenol 13.8 per cent, and that of 2-iodotyrosine from m-iodophenol 9.8 per cent.
2) Rate of synthesis of halogenated tyrosines by β-tyrosinase was slower than that of tyrosine and the rates were decreased in the order of chlorine, bromine and iodine, that is, by increasing the atomic radius. Relative rate of 2-chlorotyrosine synthesis was determined to be 28.2, that of 2-bromotyrosine to be 8.13, and that of 2-iodotyrosine to be 0.98, respectively, against 100 of tyrosine. How-ever 3-iodotyrosine was not synthesized by the enzyme.
3) The relative rate of 2-chlorotyrosine hydrolysis by β-tyrosinase was 70.7, that of 2-bromotyrosine was 39.0, and that of 2-iodotyrosine was 12.6 against 100 of tyrosine, respectively. The rate of hydrolysis appeared to be decreased in the order of chlorine, bromine and iodine, that is, by increasing the atomic radius or by decreasing the electronegativity. But 3-iodotyrosine was not hydrolyzed by the enzyme.
4) The activity of β-tyrosinase was inhibited by phenol markedly. Of the halogenated phenols, ο-, or m-chlorophenol and ο-bromophenol gave marked inhibition on the enzyme action, however inhibition by iodophenol was not strong. Plotting by Lineweaver-Burk method, a mixed-type inhibition by m-chlorophenol was observed and its Ki value was found to be 5.46×10^(-4)M.
5) During the synthesizing reaction of 2-bromotyrosine by the enzyme, sequential addition of substrate which was m-bromophsnol with time intervals and in a small amount resulted in better yield of the product.
6) The halogenated tyrosines which were produced by β-tyrosinase from pyruvate, ammonia and m-halogenated phenols were analysed to determine their molecular structures by element analysis, NMR spectroscopy, mass spectrometry, and IR spectroscopy. The result indicated that they were 2-chloro-L-tyrosine, 2-bromo-L-tyrosine, and 2-iodo-L-tyrosine, respectively.
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