Studies on myo-inositol-1-phosphate synthase from Lilium longiflorum pollen, Neurospora crassa and bovine testis Further evidence that a classical aldolase step is not utilized

Abstract

myo-Inositol-1-phosphate synthase (1 l- myo-inositol-1-phosphate lyase (isomerizing), EC 5.5.1.4.) preparations purified from the pollen of Lilium longiflorum, and from Neurospora crassa have been incubated with d-[5- 18O]-glucose -6-P and the myo-inositol which was formed was analyzed for retention of 18O. In each case, the isotope of oxygen was incorporated into the inositol without loss. If a Schiff base had formed at the 5-position of the [ 18O]glucose -6-P , the isotope should have been released to the incubation medium. Supporting evidence that no Schiff base is formed at the 5-position, or at any other position, was obtained by incubation of the enzymes in media enriched with H 2 18O. If a Schiff base were formed and hydrolyzed, the regenerated carbonyl should become enriched to the level of the medium as reflected by the isotope content of the inositol product. In no case did the product inositol have this degree of enrichment. These data exclude consideration of a Class I aldolase mechanism for these enzymes. The Lilium enzyme is unaffected by EDTA to a concentration of 100 mM. The bovine enzyme is similarly uninhibited by EDTA to a concentration of 50 mM. The Neurospora enzyme has previously been shown to be inhibited by these levels of EDTA and to be activated 2-fold by 2 mM Mg 2+, however, extensive dialysis against EDTA does not eliminate the metal independent activity of the enzyme. In the present study, we have found that divalent metals show a range of stimulation/inhibition with the Lilium and the bovine enzymes, however, neither enzyme is dependent on the metals. Thus, we suggest that these enzymes are not Class II aldolase enzymes either. It is, therefore, possible that the myo-inositol-1-phosphate synthase from these species has an aldol step in the enzymatic pathway which is of neither classical aldolase type.