SUBSTRATE-SPECIFICITY OF DIMETHYL-SULFOXI DE REDUCTASE AS PROVED BY MICELLAR ELECTROKINETIC CHROMATOGRAPHY

Micellar electrokinetic chromatography (MEKC) was applied to study the substrate specificity of dimethyl sulfoxide (DMSO) reductase from a p hotodenitrifier, Rhodobacter sphaeroides f. sp. denitrificans. Simple, direct, and accurate detection and quantification of enzymatically pr oduced compounds from the reaction mixture containing the reductase an d substrate were achieved by the MEKC assay system. This assay was sho wn to be quite effective for determination of the catalytic parameter values such as K-m and V-max. Seven pyridyl N-oxide derivatives and ad enosine N-oxide were chosen as possible novel substrates for DMSO redu ctase. Each N-oxide was incubated for an appropriate time with DMSO re ductase and its reductant in the reaction mixture. After incubation, t he mixture was directly introduced into the capillary for MEKC analysi s by which enzymatically deoxygenated compounds were monitored. Conseq uently, the eight N-oxides were proven to be suitable substrates for t he reductase. The values of K-m and V-max of the N-oxides reduction ac tivities were much larger than those of DMSO reduction activity. The V -max value of adenosine N-oxide reduction was about ten-fold higher th an those of pyridyl N-oxide derivatives reduction activities. In concl usion, MEKC was suitable for the evaluation of kinetic parameters in e nzymatic reaction. In addition, it was revealed that DMSO reductase ha s broad substrate specificity and is capable of reducing various N-oxi des.