For over 40 years, the widely accepted model for the Krebs cycle in animals assumes that GTP is produced by the succinyl-CoA synthetase reaction, which is then used to produce ATP by the transphosphorylation catalyzed by NDP kinase. Recently published evidence (Journal of Biological Chemistry 273 (1998), pp.27573-27579 and 27580-27586) demonstrates that higher eucaryotes possess the genes to express an ATP-specific succinyl-CoA synthetase (A-SCS) as well as the long-known GTP-specific succinyl-CoA synthetase (G-SCS). The two enzymes use the same type of a-subunit, but have different b-subunits that impose nucleotide specificity. The evidence also indicates that A-SCS and G-SCS are differentially expressed in various tissues. These results suggest that some cells use G-SCS to generate a mitochondrial pool of GTP that is used for purposes other than augmenting the ATP produced by oxidative phosphorylation. The overall goal of this project is to understand the metabolic implications of either enzyme being found in a particular tissue. This goal will be met by addressing the following objectives, using the mouse as a model: (1) Determine the tissue and cellular distribution of each SCS isoform. (2) Determine the genetic elements that control tissue and cellular expression by characterizing the promoter for the gene encoding the b-subunit in each type of SCS. (3) Determine the factors which alter expression of A-SCS and G-SCS. Work on these three objectives will use PCR-based methods and gene-walking techniques to obtain the sequences of the promoters, reporter assays to define the structures and properties of the promoters, Northern and Western blots to quantitate RNA and protein, immunohistochemical techniques to localize each enzyme in various tissues, and HPLC-based assays of enzyme activity to further determine tissue and cellular distribution of the enzymes. Antipeptide antibodies will be made to each of the subunits of the two enzymes for use in Western blots and immunohistochemistry. Changes in cellular and tissue expression of the enzymes will be assessed as a function of development, experimental diabetes, and chemically-induced porphyria. A fourth objective is to understand the structural basis of nucleotide specificity by locating the residues which are responsible for determining nucleotide specificity. This will be accomplished through molecular modeling and site-directed mutagenesis. The anticipated results will lead to new insights into the advantages and consequences of generating ATP vs. GTP at the substrate phosphorylation level in the Krebs cycle. Generation of GTP may provide a distinct pool of high-energy phosphate that is used for purposes either within or outside of mitochondria that cannot be met by the ATP pool formed by oxidative phosphorylation.