Characterization of Human UDP-Glucose Dehydrogenase Reveals Critical Catalytic Roles for Lysine 220 and Aspartate 280

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• 作者：Katherine E. Easley ; Brandi J. Sommer ; Gina Boanca ; Joseph J. Barycki ; Melanie A. Simpson
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：January 16, 2007
• 年：2007
• 卷：46
• 期：2
• 页码：369 - 378
• 全文大小：621K
• 年卷期：v.46,no.2(January 16, 2007)
• ISSN：1520-4995

文摘

Human UDP-glucose dehydrogenase (UGDH) is a homohexameric enzyme that catalyzes twosuccessive oxidations of UDP-glucose to yield UDP-glucuronic acid, an essential precursor for matrixpolysaccharide and proteoglycan synthesis. We previously used crystal coordinates for Streptococcuspyogenes UGDH to generate a model of the human enzyme active site. In the studies reported here, wehave used this model to identify three putative active site residues: lysine 220, aspartate 280, and lysine339. Each residue was site-specifically mutagenized to evaluate its importance for catalytic activity andmaintenance of hexameric quaternary structure. Alteration of lysine 220 to alanine, histidine, or argininesignificantly impaired enzyme function. Assaying activity over longer time courses revealed a plateauafter reduction of a single equivalent of NAD⁺ in the alanine and histidine mutants, whereas turnovercontinued in the arginine mutant. Thus, one role of this lysine may be to stabilize anionic transition statesduring substrate conversion. Mutation of aspartate 280 to asparagine was also severely detrimental tocatalysis. The relative position of this residue within the active site and dependence of function on acidiccharacter point toward a critical role for aspartate 280 in activation of the substrate and the catalyticcysteine. Finally, changing lysine 339 to alanine yielded the wild-type V_max, but a 165-fold decrease inaffinity for UDP-glucose. Interestingly, gel filtration of this substrate-binding mutant also determined itwas a dimer, indicating that hexameric quaternary structure is not critical for catalysis. Collectively, thisanalysis has provided novel insights into the complex catalytic mechanism of UGDH.