SEQUENCE AND FUNCTIONAL ANALYSIS OF TRANSCARBOXYLASE

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The research described in this proposal centers on the multisubunit enzyme, transcarboxylase from Propionibacterium shermanii. Transcarboxylase is a well characterized complex biotin-containing enzyme composed of 30 polypeptides of 3 different types; 12 1.3SE biotinyl subunits, 6 5SE dimeric outer keto acid site subunits and 1 12SH hexameric central CoA ester site subunit. The reaction catalyzed by each subunit is understood and the subunits can be dissociated and reassembled to form active enzyme. Although there is a general understanding of the mechanism of catalysis and subunit interaction, the details are just now becoming experimentally approachable. The amino acid sequence of the 1.3SE subunit is known which has permitted the isolation of several small peptides which mimic some properties of in vivo subunit interactions. These peptides have implicated the termini and the region adjacent to the biocytin of the 1.3SE subunit as being involved in binding of the outer and central subunits together and in orienting the biotin so it can serve as a carboxyl carrier. We have cloned the genes for the 1.3SE subunit and for the 5SE monomer and have expressed the former at high levels in E. coli. The expressed 1.3SE subunit is biotinated by the E. coli biotinyl synthetase and is biologically active. We will use this clone to produce a number of variant subunits whose properties will be biochemically examined. In addition, we will deduce the amino acid sequence of the 5SE monomer from its DNA and clone and sequence the 12SH monomer gene. We will construct five 1.3SE subunit variants in order to test some of the hypotheses concerning subunit function derived from peptide studies. The advantage of this approach is that alterations can be accomplished while maintaining most or all of the full length of the subunit. Specifically, we will use restriction endonuclease sites within the 1.3SE gene to produce subunits truncated on each terminus, Bal 31 to generate a series of short terminal deletions and site directed mutagenesis techniques to introduce point mutations in the vicinity of the biocytin. Each variant will be tested for its ability to be biotinated, bind outer and central subunits, function as a carboxyl carrier. Depending on the results of these studies, further modifications of the variant genes will be generated, expressed in E. coli and tested. Such mutant proteins will more closely delineate the regions of the 1.3SE peptide which are important for subunit interactions and proper enzymatic function.