SUBUNITS AND QUATERNARY STRUCTURE OF TRANSCARBOXYLASE

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This investigation is a continuation of studies of the subunit-subunit interactions of the biotin enzyme, transcarboxylase. The enzyme is made up of a hexameric central subunit which appears to have cylindrical shape. Six dimeric outer subunits are attached at the opposite faces of this central subunit, 3 at each face. The linkage of the outer to the central subunit is via 12 biotinyl subunits, 2 per outer dimeric subunit. The reaction catalyzed is CH3CH(COO-)COSCoA+CH3COCOO-CH3CH2COSCoA+OOCCH2COCOO. The CoA ester sites are on the central subunit and the keto acid sites on the outer subunits and the biotin serves as a carboxyl carrier between these sites. On the central subunit, there are 12 sites through which the outer subunits are attached via a portion of the biotinyl subunit, also 12 sites for binding of another part of the biotinyl subunit for orientation of the biotin for the carboxyl transfer, and in addition 12 CoA ester sites. Thus, each of the 6 polypeptides of the central subunit has dual sites for the 3 separate functions. The aims are to determine which portions of the biotinyl subunit are involved in its various functions. This will be accomplished by chemical synthesis of selected parts of the sequence of the biotinyl subunit and investigation of their individual binding and catalytic properties. The complementary sequences of the outside and central subunit which are involved in the subunit-subunit interaction will be investigated by cross-linking them with the biotinyl subunit. The structure of the enzyme will be investigated by X-ray crystallography and by electron microscopy of crystals of the enzyme and its subunits. The long term goal is to gain understanding of how complex enzymes in general are assembled and how the subunits interact in catalysis. Biotin enzymes have a central role in carbohydrate and lipid metabolism and a number of serious clinical syndromes arise when these enzymes are deficient. The present study contributes to understanding of the role of biotin enzymes in general.