A procedure is described for the purification of the enzyme indol-3-ylacetylglucose:myo-inositol indol-3-ylacetyltransferase (IAA-myo-inositol synthase). This enzyme catalyzes the transfer of indol-3-ylacetate from 1-0-indol-3-ylacetyl-0-d-glucose to myoinositol to form indol-3-ylacetyl-myo-inositol and glucose. A hexokinase or glucose oxidase based assay system is described. The enzyme has been purified approximately 16,000-fold, has an isoelectric point of pH 6.1 and yields three catalytically inactive bands upon acrylamide gel electrophoresis of the native protein.The enzyme shows maximum transferase activity with myo-inositol but shows some transferase activity with scyllo-inositol and myo-inosose-2. No transfer of IAA occurs with myo-inositol-dgalactopyranose, cyclohexanol, mannitol, or glycerol as acyl acceptor. The affinity of the enzyme for 1-0-indol-3-ylacetyl-ft-dglucose is, Km = 30 micromolar, and for myo-inositol is, Km = 4 millimolar. The enzyme does not catalyze the exchange incorporation of glucose into IAA-glucose indicating the reaction mechanism involves binding of IAA glucose to the enzyme with subsequent hydrolytic cleavage of the acyl moiety by the hydroxyl of myo-inositol to form IAA myo-inositol ester.This work deals with the partial purification and characterization of indol-3-ylacetylglucose:myo-inositol indol-3-ylacetyltransferase (IAInos synthase).3 The enzyme catalyzes the reaction between -0-indol-3-ylacetyl-fl-D-glucose and myoinositol to form indol-3-ylacetyl-myo-inositol and glucose according to the following equation:The reaction is important because mature kernels of Zea mays sweet corn contain 99% of their endogenous IAA as ester conjugates and less than 1% as the free acid (8). half of the esters are conjugates of IAA and myo-inositol or IAA and myo-inositol glycosides (8). The kernels contain small amounts of the isomeric conjugates of IAA and glucose, 2-0, 4-0, and 6-0-IAGlu (13). 1-0-IAGlu can be demonstrated to occur naturally in small amounts if precautions are used to prevent acyl migration (J Cohen, personal communication). An IAA glucose conjugate was the first IAA ester conjugate to be isolated from plants and was found following application of labeled IAA to plants (21,35).1-0-IAGlu is an acyl alkyl acetal4 and is chemically distinct, from an IAA ester of glucose. In aqueous solution, especially at a pH of 7 or above, the IAA moiety migrates to the position farthest from the carbonyl, that is, the 4-0 and 6-0 position. Owing to this facile acyl migration (1 1, 13,19,20,[25][26][27]) the bond energy of the acyl alkyl acetal is not readily determined but the equilibrium of the reaction:is of the order of Keq = 10-' (27) thus placing the free energy of hydrolysis of IAA glucose at about 1400 calories above that ofthe phosphato glucose bond of UDPG and many thousands of calories above that of an ester bond.These observations concerning energetics may explain the thermodynamics of the manner in which liquid endosperm of maize can contain 0.1 mm IAA ester. Once 1-0...