Characterization of human UDP-glucose dehydrogenase. CYS-276 is required for the second of two successive oxidations.
UDP-glucose dehydrogenase (UGDH) catalyzes two oxidations of UDP-glucose to yield UDP-glucuronic acid. Pathological overproduction of extracellular matrix components may be linked to the availability of UDP-glucuronic acid; therefore UGDH is an intriguing therapeutic target. Specific inhibition of human UGDH requires detailed knowledge of its catalytic mechanism, which has not been characterized. In this report, we have cloned, expressed, and affinity-purified the human enzyme and determined its steady state kinetic parameters. The human enzyme is active as a hexamer with values for Km and Vmax that agree well with those reported for a bovine homolog. We used crystal coordinates for Streptococcus pyogenes UGDH in complex with NAD+ cofactor and UDP-glucose substrate to generate a model of the enzyme active site. Based on this model, we selected Cys-276 and Lys-279 as likely catalytic residues and converted them to serine and alanine, respectively. Enzymatic activity of C276S and K279A point mutants was not measurable under normal assay conditions. Rate constants measured over several hours demonstrated that K279A continued to turn over, although 250-fold more slowly than wild type enzyme. C276S, however, performed only a single round of oxidation, indicating that it is essential for the second oxidation. This result is consistent with the postulated role of Cys-276 as a catalytic residue and supports its position in the reaction mechanism for the human enzyme. Lys-279 is likely to have a role in positioning active site residues and in maintaining the hexameric quaternary structure.
Sommer
Brandi J
BJ
Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588-0664, USA.
Barycki
Joseph J
JJ
Simpson
Melanie A
MA
D001665:Binding Sites; D020134:Catalytic Domain / Q000235:genetics; D003545:Cysteine; D006801:Humans; D008239:Lysine; D010084:Oxidation-Reduction; D011487:Protein Conformation; D013297:Streptococcus pyogenes; D013329:Structure-Activity Relationship; D014533:Uridine Diphosphate Glucose Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D014533:Uridine Diphosphate Glucose Dehydrogenase; D008239:Lysine; D003545:Cysteine