Glycogen storage disease type 1 (GSD-1) is caused by a deficiency in the activity of glucose-6-phosphatase (G6Pase), an endoplasmic reticulum (ER) associated enzyme. The disease presents with both clinical and biochemical heterogeneity consistent with the existence of two major subgroups, GSD-1a and GSD-1b. GSD-1a, the most prevalent form, is caused by deleterious mutations in the G6Pase gene. We have recently resolved the topology of G6Pase and shown that it contains an odd number of transmembrane helices with the N-terminus localized in the ER lumen and the C-terminus in the cytoplasm. This supports a nine-transmembrane helical model with the enzyme's active site facing the lumen. The topology of G6Pase was further explored by characterizing the three potential Asn-linked glycosylation sites (N96TS, N203AS, and N276SS) in G6Pase. We showed that N96, predicted by the nine-transmembrane model to be located in a 37-residue luminal loop and would be a potential acceptor for oligosaccharides, was the only site that is glycosylated, further supporting the nine transmembrane domain model.It has been proposed that GSD-1b is caused by a deficiency in microsomal glucose-6-phosphate (G6P) transport. By linkage analysis, we have mapped the GSD-1b locus to chromosome 11q23 and a cDNA encoding a microsomal transmembrane protein has been identified. We showed that this cDNA mapped to chromosome 11q23, thus it is a strong candidate for GSD-1b. The crystal structure of the E. coli methionine adenosyltransferase (MAT) has been solved, demonstrating that the active site of the enzyme is at the interface between two identical subunits. To investigate the catalysis of human hepatic MAT, we constructed a series of mutations of the amino acids proposed to be in the active site and one of the putative ATP binding sites. Each of these mutations reduced enzymatic activity significantly, suggesting that the human and E. coli enzyme are arrayed in a similar fashion. We have combined mutational with three dimensional analyses to propose a model for the active site of human MAT.
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