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KMID : 0578319920020010109
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Molecules and Cells
1992 Volume.2 No. 1 p.109 ~ p.114
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Site-directed Mutagenesis of Arginine-178 of thymidylate Synthase from Lactobacillus casei
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Cho Wung-Woo
Choi Soo-Young
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Abstract
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X-ray structural studies have previously shown that Arg-178 of thymidylate synthase interacts with bound inorganic phosphate or with the 5´-phosphate of the bound substrate dUMP. The importance of Arg-178 to the structure/function of thymidylate synthase is also indicated by its complete conservation among the 17 thymidylate synthases thus far sequenced. In the present work, cassette mutagenesis has been performed for Arg-178 of Lactobacillus casei thymidylate synthase. Eleven amino acid substitutions have been obtained for Arg-178. Methods have been developed for determination of functional and phenotypical characteristics for each of the newly synthesized mutant proteins. Functionally acceptable substitutions were defined by genetic complementation of thymidylate synthase deficient cells and further characterized by determination of specific activity. Evaluation of kinetic parameters of the mutants was performed in crude extract using 5-fluoro-2´-deoxyuridylate as an active site titrant. Analysis of the mutants by genetic complementation indicates that thymidylate synthase can tolerate a number of amino acid substitutions at that position and shows that Arg-178 is not strictly required for catalytic activity.
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KEYWORD
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