SUBSTRATE-SPECIFICITY OF DIMETHYL-SULFOXIDE 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 photodenitrifier, Rhodobacter sphaeroides f. sp. denitrificans. Simple, direct, and accurate detection and quantification of enzymatically produced compounds from the reaction mixture containing the reductase and substrate were achieved by the MEKC assay system. This assay was shown to be quite effective for determination of the catalytic parameter values such as K-m and V-max. Seven pyridyl N-oxide derivatives and adenosine N-oxide were chosen as possible novel substrates for DMSO reductase. Each N-oxide was incubated for an appropriate time with DMSO reductase and its reductant in the reaction mixture. After incubation, the mixture was directly introduced into the capillary for MEKC analysis by which enzymatically deoxygenated compounds were monitored. Consequently, the eight N-oxides were proven to be suitable substrates for the reductase. The values of K-m and V-max of the N-oxides reduction activities were much larger than those of DMSO reduction activity. The V-max value of adenosine N-oxide reduction was about ten-fold higher than those of pyridyl N-oxide derivatives reduction activities. In conclusion, MEKC was suitable for the evaluation of kinetic parameters in enzymatic reaction. In addition, it was revealed that DMSO reductase has broad substrate specificity and is capable of reducing various N-oxides.