Unusual sugar nucleotide recognition elements of mesophilic vs. thermophilic glycogen synthases.
The glycogen synthase found in Pyrococcus furiosus is a hyperthermophilic biocatalyst that transfers the glucose portion of nucleotide-diphosphoglucose onto a growing carbohydrate biopolymer chain at 80 degrees C. In contrast to the mesophilic rabbit muscle glycogen synthase, the biocatalyst from P. furiosus possesses unusually broad nucleotide tolerance. The enzyme accepts all four common glucose-containing nucleotide-diphosphosugars: ADP-glucose, GDP-glucose, dTDP-glucose, and UDP-glucose. Using an electrospray ionization-mass spectroscopy (ESI-MS) assay, we determined the K(M) and Vmax for GDP-glucose to be 3.9 +/- 0.6 mM and 0.243 +/- 0.009 mM/min, and for dTDP-glucose to be 4.0 +/- 0.5 mM and 0.216 +/- 0.008 mM/min. A related nucleotide sugar, UDP-galactose, was not a reactive substrate, but was instead a competitive inhibitor with a Ki of 17 +/- 2 mM. The glycogen synthase from P. furiosus was shown not to have phosphorylase activity. The DeltaDeltaG of substrate binding was compared between the mesophilic rabbit muscle and the hyperthermophilic P. furiosus glycogen synthase to dissect any differences in sugar nucleotide recognition strategies at elevated temperatures. Both biocatalysts were shown to gain most of their substrate affinity through electrostatic interactions between the enzyme and the alpha-phosphate.
Zea
Corbin J
CJ
Department of Chemistry and the Plant Sciences Institute, Gilman Hall, Iowa State University, Ames, IA 50011-3111, USA.
Pohl
Nicola L
NL
D000818:Animals; D001665:Binding Sites; D006006:Glycogen Synthase / Q000737:chemistry* / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D020027:Pyrococcus furiosus / Q000201:enzymology; D011817:Rabbits
D006006:Glycogen Synthase