Characterization of human UDP-glucose dehydrogenase reveals critical catalytic roles for lysine 220 and aspartate 280

Katherine E. Easley, Brandi J. Sommer, Gina Boanca, Joseph J Barycki, Melanie A Simpson

Research output: Contribution to journalArticle

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Abstract

Human UDP-glucose dehydrogenase (UGDH) is a homohexameric enzyme that catalyzes two successive oxidations of UDP-glucose to yield UDP-glucuronic acid, an essential precursor for matrix polysaccharide and proteoglycan synthesis. We previously used crystal coordinates for Streptococcus pyogenes UGDH to generate a model of the human enzyme active site. In the studies reported here, we have used this model to identify three putative active site residues: lysine 220, aspartate 280, and lysine 339. Each residue was site-specifically mutagenized to evaluate its importance for catalytic activity and maintenance of hexameric quaternary structure. Alteration of lysine 220 to alanine, histidine, or arginine significantly impaired enzyme function. Assaying activity over longer time courses revealed a plateau after reduction of a single equivalent of NAD+ in the alanine and histidine mutants, whereas turnover continued in the arginine mutant. Thus, one role of this lysine may be to stabilize anionic transition states during substrate conversion. Mutation of aspartate 280 to asparagine was also severely detrimental to catalysis. The relative position of this residue within the active site and dependence of function on acidic character point toward a critical role for aspartate 280 in activation of the substrate and the catalytic cysteine. Finally, changing lysine 339 to alanine yielded the wild-type Vmax, but a 165-fold decrease in affinity for UDP-glucose. Interestingly, gel filtration of this substrate-binding mutant also determined it was a dimer, indicating that hexameric quaternary structure is not critical for catalysis. Collectively, this analysis has provided novel insights into the complex catalytic mechanism of UGDH.

Original languageEnglish (US)
Pages (from-to)369-378
Number of pages10
JournalBiochemistry
Volume46
Issue number2
DOIs
StatePublished - Jan 16 2007

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Uridine Diphosphate Glucose Dehydrogenase
Aspartic Acid
Lysine
Alanine
Uridine Diphosphate Glucose
Catalytic Domain
Catalysis
Histidine
Arginine
Substrates
Enzymes
Uridine Diphosphate Glucuronic Acid
Streptococcus pyogenes
Asparagine
Proteoglycans
Dimers
NAD
Gel Chromatography
Polysaccharides
Cysteine

ASJC Scopus subject areas

  • Biochemistry

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Characterization of human UDP-glucose dehydrogenase reveals critical catalytic roles for lysine 220 and aspartate 280. / Easley, Katherine E.; Sommer, Brandi J.; Boanca, Gina; Barycki, Joseph J; Simpson, Melanie A.

In: Biochemistry, Vol. 46, No. 2, 16.01.2007, p. 369-378.

Research output: Contribution to journalArticle

Easley, Katherine E. ; Sommer, Brandi J. ; Boanca, Gina ; Barycki, Joseph J ; Simpson, Melanie A. / Characterization of human UDP-glucose dehydrogenase reveals critical catalytic roles for lysine 220 and aspartate 280. In: Biochemistry. 2007 ; Vol. 46, No. 2. pp. 369-378.
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abstract = "Human UDP-glucose dehydrogenase (UGDH) is a homohexameric enzyme that catalyzes two successive oxidations of UDP-glucose to yield UDP-glucuronic acid, an essential precursor for matrix polysaccharide and proteoglycan synthesis. We previously used crystal coordinates for Streptococcus pyogenes UGDH to generate a model of the human enzyme active site. In the studies reported here, we have used this model to identify three putative active site residues: lysine 220, aspartate 280, and lysine 339. Each residue was site-specifically mutagenized to evaluate its importance for catalytic activity and maintenance of hexameric quaternary structure. Alteration of lysine 220 to alanine, histidine, or arginine significantly impaired enzyme function. Assaying activity over longer time courses revealed a plateau after reduction of a single equivalent of NAD+ in the alanine and histidine mutants, whereas turnover continued in the arginine mutant. Thus, one role of this lysine may be to stabilize anionic transition states during substrate conversion. Mutation of aspartate 280 to asparagine was also severely detrimental to catalysis. The relative position of this residue within the active site and dependence of function on acidic character point toward a critical role for aspartate 280 in activation of the substrate and the catalytic cysteine. Finally, changing lysine 339 to alanine yielded the wild-type Vmax, but a 165-fold decrease in affinity for UDP-glucose. Interestingly, gel filtration of this substrate-binding mutant also determined it was a dimer, indicating that hexameric quaternary structure is not critical for catalysis. Collectively, this analysis has provided novel insights into the complex catalytic mechanism of UGDH.",
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