The proposed investigation extends ongoing studies on pyridoxal phosphate (PLP)-dependent enzymes of known tertiary structure. Work during the past period of support led to the production of cDNA clones for aspartate transaminase (AAT) and their expression as intact precursor for mitochondrial AAT in E. coli. This precursor (pmAAT) was produced in large quantities and is the first purified precursor of a mitochondrial-targeted protein available as an isolated, stable protein. Furthermore, the amino terminal region of the mature enzyme has been identified as important for the PLP binding environment. Recent work also contributed to the development of a variety of spectroscopic and other physical techniques as tools for analyzing individual PLP regions and their proximity to protein binding sites and vice versa. These tools, along with specific anti-PLP antibodies and selected inhibitors of this well-characterized enzyme, will be used for the new studies. The proposed investigations are: 1. To study the structural changes associated with the conversion of the first isolated mitochondrial protein precursor (pmAAT) into a mature PLP- dependent enzyme (mAAT). 2. To use site-specific mutagenesis of pmAAT to assess the roles of some selected individual residues on PLP binding and protein stability, and to relate the changes in precursor structure in solution as induced by the introduction of alterations in this dimeric and coenzyme requiring protein to the rate or extent of its translocation into mitochondria. 3. To determine the minimum protein size needed to maintain affinity for PLP, retention of individual enzymatic functions, and possible import into mitochondria. The impact of these findings can have consequences beyond the PLP field, as they impinge into three fundamental biological problems: (a) Molecular structure for proteins of much primary sequence analogy (precursor/mature protein); (b) Effects of the presequence peptide on overall protein structure and properties of precursors; and (c) How precursors are recognized, imported and processed by mitochondria.
