STEREOCHEMISTRY AND MECHANISMS OF ENZYME REACTIONS

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Further studies of enzymes catalyzing allylic and propargylic rearrangements are proposed. Beta-Hydroxydecanolythioester dehydrase (E. coli) is crucial to the biosynthesis of unsaturated fatty acids under anaerobic conditions, catalyzing interconversion of (R)-3-hydroxydecanoate, E-2-decenoate, and Z-3-decenoate thioesters. A histidine residue is apparently the sole active site base/conjugate acid, although there is inconclusive evidence for an essential tyrosine. Dehydrase is irreversibly inactivated by the substrate analog 3-decynol-NAC (NAC = N-acetycysteamine), via 2,3-decadienoyl-NAC. The allenic thioester alkylates the essential histidine. An allene-acetylene isomerase (AAI) isolated from hog liver interconverts 2,3- and 3,4-enoyl thioesters, though it functions more efficiently with 3-ynoyl and 2,3-dienoyl thioester substrates. AAI is unaffected by 2,3-decadienoyl-NAC. Proposed studies on dehydrase and AAI include: (a) determination by 15N NMR) of which (nonequivalent) imidazole nitrogen of dehydrase becomes attached to C-3 of the inactivator; (b) application of bifunctional inactivators, bearing both a 3,4-acetylenic carbonyl function and a second reactive (or latently reactive) functional group, as a probe of a different region of the active site; (c) crystallization of dehydrase and preliminary x-ray crystallographic studies; (d) design, synthesis, and implementation of novel potential inactivators of AAI; (e) isolation and characterization of AAI from an alternative (hopefully bacterial) source, in order to (ultimately) amplify the yield of AAI and to facilitate its isolation. The significance of this project is multifold: (a) to identify structural features, particularly at the active site, that control the function of the enzyme that is unique, and indispensible, to the biosynthesis of unsaturated fatty acids under anaerobic conditions, and in doing so, (b) to develop methods for multiple attachment of inactivators to target enzymes; (c) to derive useful information regarding the active site structure and mechanism of action of AAI, an unusual and possibly metabolically important enzyme for which there is little mechanistic information; (d) to further demonstrate the utility and versatility of fluorine-substituted compounds as affinity labels, "suicide " substrates, and competitive enzyme inhibitors; (e) to develop an alternative source of AAI.