Ross Kerry L, Davis Charity N, Fridovich-Keil Judith L
Department of Human Genetics, Emory University School of Medicine, Emory University, Atlanta, GA 30322, USA.
Mol Genet Metab. 2004 Sep-Oct;83(1-2):103-16. doi: 10.1016/j.ymgme.2004.07.005.
The metabolism of galactose via enzymes of the Leloir pathway: galactokinase, galactose-1-P uridylyltransferase, and UDP galactose-4'-epimerase, is a process that has been conserved from Escherichia coli through humans. Impairment of this pathway in patients results in the disease galactosemia. Despite decades of study, the underlying pathophysiology in galactosemia remains unknown. Here we have defined the functional and metabolic implications of impaired galactose metabolism in yeast, by asking two questions: (1) What is the impact of loss of each of the three Leloir enzymes on the ability of cells to metabolize galactose, and on their sensitivity to galactose, and (2) what is the relationship between gal-1P and galactose-sensitivity in yeast? Our results demonstrate that only transferase-null cells are able to deplete their medium of galactose; deletion of kinase or epimerase halts this process. In contrast, only kinase-null cultures grow well in glycerol/ethanol medium despite the addition of galactose; both transferase and epimerase-null yeast arrest growth under these conditions. Indeed, epimerase-null yeast arrest growth at galactose concentrations 10-fold lower than do their transferase-null counterparts. Secondary deletion of kinase relieves growth arrest in both strains. Finally, rather than a continuous relationship between gal-1P and growth arrest, we observed a threshold level of gal-1P (approximately 10 nmol/mg cell DM) above which both transferase-null and epimerase-null cultures could not grow. These results both confirm and significantly extend prior knowledge of galactose metabolism in yeast, and set the stage for future studies into the mediators and mechanism of Leloir-impaired galactose sensitivity in eukaryotes.
通过Leloir途径的酶（半乳糖激酶、1-磷酸半乳糖尿苷酰转移酶和UDP半乳糖-4'-表异构酶）进行的半乳糖代谢，是一个从大肠杆菌到人类都保守的过程。患者体内该途径受损会导致半乳糖血症。尽管经过了数十年的研究，但半乳糖血症的潜在病理生理学仍然未知。在这里，我们通过提出两个问题来确定酵母中半乳糖代谢受损的功能和代谢影响：（1）三种Leloir酶中每种酶的缺失对细胞代谢半乳糖的能力及其对半乳糖的敏感性有何影响，以及（2）酵母中1-磷酸半乳糖（gal-1P）与半乳糖敏感性之间的关系是什么？我们的结果表明，只有缺失转移酶的细胞能够耗尽培养基中的半乳糖；激酶或表异构酶的缺失会阻止这一过程。相比之下，尽管添加了半乳糖，但只有缺失激酶的培养物在甘油/乙醇培养基中生长良好；缺失转移酶和表异构酶的酵母在这些条件下会停止生长。事实上，缺失表异构酶的酵母在半乳糖浓度比缺失转移酶的酵母低10倍时就会停止生长。激酶的二次缺失可缓解两种菌株的生长停滞。最后，我们观察到gal-1P与生长停滞之间并非连续关系，而是存在一个阈值水平（约10 nmol/mg细胞干重），高于该水平，缺失转移酶和表异构酶的培养物都无法生长。这些结果既证实了并显著扩展了之前关于酵母中半乳糖代谢的知识，也为未来研究真核生物中Leloir途径受损导致半乳糖敏感性的介质和机制奠定了基础。
