Kinetic and structural properties of diacetyl reductase from hamster liver

Kinetic and physicochemical properties of hamster liver diacetyl reductase have been examined. The results of kinetic studies on the reduction of diacetyl and NADPH to acetoin and NADP+ suggest that the reaction follows an Ordered Bi Bi mechanism in which NADPH binds first before diacetyl. The enzyme is a tetrameric glycoprotein of single subunits of a molecular weight of 23,500 with a sedimentation coefficient of 6.0S. The enzyme does not contain Zn, Cu, or Fe. The amino acid composition revealed an unusually low proportion of proline residues (0.9%). p-Chloromercuriphenylsulfonate and phenylglyoxal inactivated the enzyme, but the presence of NADPH prevented the loss of activity due to thiol and arginine modification. The enzyme transferred the pro 4S hydrogen atom of NADPH to the substrate and the binding of the enzyme to NADPH resulted in a red shift of the ultraviolet absorption spectrum of the cofactor.