L-myo-Inosose-1 as a probable intermediate in the reaction catalyzed by myo-inositol oxygenase

Abstract

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)

myo-Inositol oxygenase: a radical new pathway for O2and C–H activation at a nonheme diiron cluster

“…An interesting possibility is that the inhibitor could have formed in the active site of the truncated protein as a consequence of derangement of the catalytic cycle by removal of the active-site-enclosing N-terminal loop. If so, the bound myo -inosose-1 could be an intermediate in the normal catalytic pathway (as originally proposed by Hamilton and co-workers61) or a breakdown product thereof. Mechanistic studies on the truncated protein could, therefore, potentially be informative.…”

“…31 In the first, which we designate the “hydroperoxylation” path, formation of the new C1-O bond precedes the bond-cleavage steps. The intermediate in this pathway, (1-hydroperoxy)- myo -inositol, was first envisaged by Hamilton and co-workers in the 1980s, who cited chemical precedent that it would break down to DG 61. In the second class, designated the “hydroxylation” path, the (hydroperoxo)diiron(III/III) intermediate decays by attack of the C1 radical on the distal O-atom of the hydroperoxide, leading simultaneously to hydroxylation of C1 and cleavage of the O-O bond.…”

“…The most striking difference, however, is the assignment by the authors of the bound species as myo -inosose-1 (the 2-electron-oxidized 1-ketone form of MI), despite the fact that only MI was added in the crystallization. The authors suggested that this known MIOX inhibitor61 could have been formed during the long (~ 2-month) incubation required for crystal growth or could have been a contaminant in the commercial MI stock that they employed. An interesting possibility is that the inhibitor could have formed in the active site of the truncated protein as a consequence of derangement of the catalytic cycle by removal of the active-site-enclosing N-terminal loop.…”

“…Thus, the hydroxylation pathway would seem to be more likely than the hydroperoxylation pathway, if the active complex is not significantly different from the published structures. However, several other pathways from either the coordinated gem -diolate or a ketone intermediate61 can also be envisaged.…”

“…The intermediate in this pathway, (1-hydroperoxy)-myo-inositol, was first envisaged by Hamilton and co-workers in the 1980s, who cited chemical precedent that it would break down to DG. 61 In the second class, designated the "hydroxylation" path, the (hydroperoxo)diiron(III/III) intermediate decays by attack of the C1 radical on the distal O-atom of the hydroperoxide, leading simultaneously to hydroxylation of C1 and cleavage of the O-O bond. The resultant substrate species would be a coordinated gem-diol(ate), and the cofactor would be in the III/IV oxidation state.…”