The objective of this research is to identify the structural determinants of multienzyme complex formation and biochemical function for transcarboxylase. Transcarboxylase is a 1.2 million dalton multienzyme complex that catalyzes the transfer of CO2 from methylmalonyl coenzyme A to pyruvate, to form propionyl coenzyme A and oxaloacetate, by way of two biotin-dependent half reactions. This multienzyme complex contains three different types of subunits and a total of 30 polypeptide chains: a 396 kDa hexameric central "12S" subunit, six 116 kDa dimeric outer "5S" subunits; and twelve 12 kDa biotinylated "1.3S" linkers. X-ray crystal structures of the holo enzyme and of its largest (12S and 5S) subunits, in free form and bound to substrate or product, will be determined. The first specific aim of this work is to determine the crystal structures of 12S substrate and product complexes, by heavy atom phasing and subsequent molecular replacement methods. These structures will identify the structural determinants of substrate binding and catalysis for the first transcarboxylase half reaction. In particular, the protein residues which interact with substrate or product molecules will be revealed; the geometry and chemistry of these amino acids as well as others nearby will be analyzed in order to speculate about likely catalytic mechanism(s). The second specific aim is to determine the crystal structures of the free 12S protein, and of 12S bound to biotin, by molecular replacement methods. Comparison of these structures with the substrate and product complexes will allow identification of the conformational changes which occur upon substrate or biotin binding, and may extend the structural insight into catalytic mechanism. Finally, the third specific aim is to determine the crystal structures of the 5S, 6S, and holo enzyme forms of transcarboxylase, either free or bound to substrate, by a combination of heavy atom phasing and molecular replacement methods. The 5S structures will provide insight into the catalytic mechanism for the second transcarboxylase half reaction, by identifying amino acids which are important in substrate binding and/or catalysis. The 6S and holo enzyme structures will highlight conformational changes and interaction surfaces which are important for enzyme assembly and function.
The assembly of transcarboxylase crystal structures which are being pursued will reveal the features important in homo-oligomeric subunit assembly as well as reveal the structural features of intersubunit assembly. These results will provide a rare structural view of a multienzyme complex. Transcarboxylase has historically served as a model system, which is valuable for understanding the mechanisms for other (de)carboxylase enzymes that carry out similar chemistry.
