Studies of rat beta-methylcrotonyl-coenzyme A carboxylase
Date of Award
Doctor of Philosophy (Ph.D.)
First Committee Member
Fazal Ahmad, Committee Chair
Initial sequence analysis of a cDNA clone isolated from a rat liver cDNA library with a polyclonal anti-pyruvate carboxylase antibody suggested that this clone encoded another biotin-dependent enzyme, possibly representing $\beta$-methylcrotonyl-Coenzyme A carboxylase, the fourth biotin-dependent mammalian carboxylase of yet unknown primary structure. In order to confirm the identity of this clone, rat kidney $\beta$-methylcrotonyl-Coenzyme A carboxylase was purified to apparent homogeneity. The $\alpha$-subunit, the polypeptide which contains the bound biotin prosthetic group, was separated from the $\beta$-subunit and subjected to digestion by Endo-Lys C protease. The resulting peptides and the amino-terminus of the intact subunit were sequence analyzed. Colinearity between cDNA-deduced and chemically-determined sequence identified this clone as the $\alpha$-subunit of $\beta$-methylcrotonyl-Coenzyme A carboxylase. Additional overlapping clones produced an open reading frame representing the primary structure of the $\alpha$-subunit of $\beta$-methylcrotonyl-Coenzyme A carboxylase lacking only about 0.5 kb of its 5$\sp\prime$-terminus. The combination of chemically-determined and cDNA-deduced sequence defines the nearly complete primary structure of $\beta$-methylcrotonyl-Coenzyme A carboxylase.A comparison of $\beta$-methylcrotonyl-Coenzyme A carboxylase sequence with primary structures of other biotin enzymes and subunits has permitted identification of the location of the biotin carboxylase and the biotin carboxyl carrier protein domains on the $\alpha$-subunit of this multienzyme complex. The biotin carboxylase domain has been further dissected into five "subdomains" which have been similarly identified within all presently characterized biotin carboxylating subunits and polypeptides. In addition, inter-domain spacing analyses of all biotin enzymes and subunits possessing biotin carboxylase activity has quantitatively described a lineage of compound fusion events which may have been responsible for creating multifunctional polypeptides from ancestral unifunctional subunits.
Ditmer, Laura Susan, "Studies of rat beta-methylcrotonyl-coenzyme A carboxylase" (1993). Dissertations from ProQuest. 3106.