This is a proposal to study mitochondrial and cytosolic aspartate transaminase isozymes.
We aim to: a) understand molecular forces responsible for each protein's stability, and the characterization of structural elements associated with specific roles in each of reaction mechanisms (chemistry); b) investigate their susceptibility to endocellular protein processing events (proteases) and the mechanisms of protein passage into organelles such as mitochondria (biology); and c) manipulate chemically and genetically to perturb specific enzymatic functions, structural protein elements or those necessary for recognition for passage through membranes (molecular biology). To this end we propose to: a) utilize differential scanning and batch calorimetry, 31P and 13CNMR, as well as FTIR to analyze structural factors affecting each isozyme's stability induced as a consequence of modification by action of selected proteases or chemical manipulation b) characterize the pyrophosphatase-like activity of the apo form of the isozymes; c) study the effects of protease action or chemical modification of the isozymes on mitochondrial recognition and/or protein transport mechanisms; d) produce semisynthetic enzymes in which the 14-19 residue N-terminal portion is judiciously altered and follow the consequences of such alterations; e) search for a mechanisms of action of factors, such as polyphosphates, controlling endocellular protease processing of the isozymes; and f) isolate and express the mitochondrial isozyme's cDNA in cells to produce precursor forms of this isozyme to study the molecular properties of the precursor, its mechanism of transport into mitochondria, and process of conversion into mature isozyme.
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