CRYSTAL STRUCTURE OF A TRANSCARBOXYLASE (TC) MULTIENZYME COMPLEX

这个子项目是众多研究子项目之一

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The goal of the proposed research is to determine crystal structures of the transcarboxylase (TC) multienzyme complex and related proteins. TC is a 1.2 million dalton biotin-dependent bacterial metabolic enzyme that has long served as a model system for human biotin-dependent metabolic enzymes which have been difficult to study using biochemical and structural methods; none of the human or mammalian biotin-dependent enzymes have been crystallized. Crystal structures of TC will provide insight into mechanisms of assembly and catalysis for biotin-dependent metabolic enzymes. Intact 26S TC contains 30 polypeptide chains of three types: the catalytic 12S which forms a hexamer, the catalytic 5S which forms a dimer, and the biotinylated 1.3S which shuttles between the 12S and 5S active sites. The 26S TC can lose several 5S subunits to give the 24 polypeptide chain 18S, which is also enzymatically active. We have recently published crystal structures of the isolated 12S subunit (1) and of the isolated 5S subunit (2), in free form and as several active site complexes, determined using synchrotron radiation. While these structures provide some initial understanding of enzyme mechanism, structures of the 18S and 26S subunits are expected to be much more informative in order to understand multienzyme complex assembly and regulation, as well as to extend speculations about enzyme mechanism and the molecular basis of human metabolic disorders resulting from mutations in the human enzymes. We propose to determine crystal structures of TC 26S, 18S and 6S, which are hetero-oligomeric forms containing 3 or 2 different subunits, as well as additional structures of isolated 12S and 5S, either as active site ligand complexes or mutations designed to probe enzyme mechanism or to reproduce the effect of human mutations identified in metabolic diseases.