L-myo-inosose-1 as a probable intermediate in the reaction catalyzed by myo-inositol oxygenase.
In previous investigations, it was necessary to have Fe(II) and cysteine present in order to assay the catalytic activity of purified hog kidney myo-inositol oxygenase. In the present study it was found that, if this purified nonheme iron enzyme is slowly frozen in solution with glutathione and stored at -20 degrees C, it is fully active in the absence of activators if catalase is present to remove adventitious H2O2. With this simpler assay system it was possible to clarify the effects of several variables on the enzymic reaction. Thus, the maximum velocity is pH-dependent with a maximum around pH 9.5, but the apparent Km for myo-inositol (air atmosphere) remains constant at 5.0 mM throughout a broad pH range. The enzyme is quite specific for its substrate myo-inositol, is very sensitive to oxidants and reductants, but is not affected by a variety of complexing agents, nucleotides, sulfhydryl reagents, etc. In other experiments it was found that L-myo-inosose-1, a potential intermediate in the enzymic reaction, is a potent competitive inhibitor (Ki = 62 microM), while other inososes and a solution thought to contain D-glucodialdehyde, another potential intermediate, are weak inhibitors. Also, both a kinetic deuterium isotope effect (kH/kD = 2.1) and a tritium isotope effect (kH/kT = 7.5) are observed for the enzymic reaction when [1-2H]- and [1-3H]-myo-inositol are used as reactants. These latter results are considered strong evidence that the oxygenase reaction proceeds by a pathway involving L-myo-inosose-1 as an intermediate rather than by an alternative pathway that would have D-glucodialdehyde as the intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D003903:Deuterium; D006863:Hydrogen-Ion Concentration; D007294:Inositol / Q000031:analogs & derivatives* / Q000378:metabolism; D050562:Inositol Oxygenase; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D010105:Oxygenases / Q000378:metabolism*; D013552:Swine; D014316:Tritium
D014316:Tritium; C002183:inosose; D007294:Inositol; D003903:Deuterium; D010105:Oxygenases; D050562:Inositol Oxygenase