One of the fundamental questions in molecular biology concerns the relationship between primary amino acid sequence and the structure and function of proteins involved in cellular metabolic processes. The long-term goal of this project is to elucidate the structure/function relationships and interactions of human enzymes associated with galactosemia, a potentially lethal inborn error of metabolism that affects 1/30,000 to 1/60,000 live-born children. The proposed experiments focus on two enzymes, galactose-l-phosphate uridylyltransferase (GALT, EC 2.7.7.12) and UDP-galactose-4-epimerase (GALE, EC 5.1.3.2), both of which function in the Leloir pathway of galactose metabolism; impairment of either can result in a clinically significant disease. The human cDNAs/genes encoding both enzymes have been cloned and characterized; >50 different point mutations have been reported in the GALT alleles of transferase-deficiency patients, and 5 point mutations have been reported in the GALE alleles of epimerase-deficiency patients. Identifying a candidate mutation is far from understanding it, however. Despite recent advances, fundamental questions remain concerning the normal structures, functions, and interactions of these enzymes, and the impact(s) of naturally occurring mutations on each. The short-term goal of this project is to address these questions by achieving the following specific aims: [1] to explore the structure/function relationships of wild-type human GALT, using both yeast and mammalian cells, [2] to analyze the molecular and biochemical impact of known naturally-occurring GALT mutations derived from patients with transferase-deficiency galactosemia, [3] to analyze the molecular and biochemical impact of known naturally occurring GALE mutations derived from patients with epimerase-deficiency galactosemia, and [4] to investigate the possibility of physical and/or functional interactions between enzymes of the Leloir pathway. The results of these studies will be significant because in addition to making iterative advances in the knowledge of GALT and GALE structure/function relationships, by demonstrating and examining the subcellular sequestration of these enzymes, this work will challenge the current paradigm of their function. In addition, by providing a more accurate understanding of the functional consequences of specific patient mutations in GALT and GALE, these studies will enable both improved diagnostic and prognostic tools, and ultimately should form a rational approach to the generation of novel treatments for patients with galactosemia.
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