Hydrolysis of some chromogenic substrates and adenosine triphoshate catalyzed by phosphodiesterase-phosphomonoesterase from Fusarium moniliforme.

概要

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For comparison with known phosphodiesterases, the mode of action of phosphodiesterase-phosphomonoesterase from Fusarium moniliforme toward some key substrates was examined. The enzyme catalyzed the hydrolysis of either thymidine 3′-(4-nitrophenylphosphate) or thymidine 5′-(4-nitrophenylphosphate). The former substrate was hydrolyzed processively into thymidine, inorganic phosphate and 4-nitrophenol, whereas the latter was split into 4-nitrophenol and thymidine 5′-phosphate. The kinetic constants determined on the basis of 4-nitrophenol liberation were: Km=4.6 mmol dm−3 and kcat=115 s−1 for thymidine 3′-(4-nitrophenylphosphate); Km=0.46 mmol dm−3 and kcat=19.7 s−1 for thymidine 5′-(4-nitrophenylphosphate) at pH 5.3 and 37 °C. 4-Nitrophenyl phenylphosphonate was a good substrate for the enzyme showing kinetic constants Km=1.4 mmol dm−3 and kcat=360 s−1 at pH 5.3 and 37 °C. However, its chiral thio analogue, O-(4-nitrophenyl) phenylphosphonothioate, was hydrolyzed by the enzyme only very slowly in a stereo-nonspecific manner. The three phosphoryl groups in adenosine triphosphate was removed by the enzyme sequentially from the terminal position. These findings have made the uniqueness of Fusarium phosphodiesterase-phosphomonoesterase clearer.
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