50(P09) 好熱性細菌Bacillus stearothermopilusの野生型および点変異FPP合成酵素の基質特異性(ポスター発表の部)

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Farnesyl diphosphate (FPP)synthase [2.5.1.10] catalyzes the condensation of isopentenyl diphosphate (IPP) with dimethylallyl diphosphate (DMAPP) and with geranyl diphosphate (GPP) to produce E,E-FPP. The specificities of the wild-type and mutant FPP synthase for artificial substrates have been studied using the substrate analogs 1-7 and 9-12, which have substituents with various chain length at 4-position of DMAPP. Previous study with the analogs showed that the substrates specificities of the bacterial enzyme was very similar to those of pig liver and chicken liver enzymes. Cysteine residues in several enzymes play important roles in their catalytic function. FPP synthase from B. stearothermophilus has two cysteine residues at positions of 73 and 289. The mutant enzymes, C73S, C2895 and C73S-C289S, in which cysteine residues were replaced with serine, showed that they all have similar substrate specificities to that of the wild-type enzyme. This result also indicates that these cysteine residues in this enzyme is not important for catalytic function as reported before. The highly conserved Gln residue at position 221 in an upstream part of the putative substrate binding site was replaced with Glu. The substrate specificity of this mutant enzyme has been also studied with some DMAPP analogs. These analogs 3 and 4 were as reactive as GPP for the wild-type enzyme. However, the mutagenesis resulted in heavy decrease of activities of the analogs tested to one twentieth of activities for the wild-type enzyme. In contrast, the K_m value of the analog 3 for the mutant enzyme Q221E was comparable to that for the wild-type enzyme. G1n 221 is involved not only in the binding of allylic substrates but also in the catalysis by FPP synthase.
天然有機化合物討論会の論文
1996-09-02

50(P09) 好熱性細菌Bacillus stearothermopilusの野生型および点変異FPP合成酵素の基質特異性(ポスター発表の部)

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