General assay for sugar nucleotidyltransferases using electrospray ionization mass spectrometry.
An electrospray ionization mass spectrometry-based assay has been developed to study the class of enzymes called sugar nucleotidyltransferases that couple sugar-1-phosphates and nucleotide triphosphates to form Leloir pathway glycosyl donors. The recombinant Escherichia coli and the commercially available yeast uridine-diphosphoglucose pyrophosphorylases were used as model systems. This technique allows the simultaneous and direct detection of the substrates and products without separation and, as described, is as sensitive as traditional coupled techniques. More importantly, the assay is capable of easily measuring kinetic values and inhibition constants for a range of natural and nonnatural substrates. This new assay was used to show for the first time that the reaction of the commercially available yeast uridine-diphosphoglucose pyrophosphorylase preparation is competitively inhibited by adenosine 5'-triphosphate (ATP), an observation that indicates a single active site that accepts both uridine 5'-triphosphate and ATP substrates.
Zea
Corbin J
CJ
Department of Chemistry and the Plant Sciences Institute, Iowa State University, Ames, IA 50011, USA.
Pohl
Nicola L
NL
D000255:Adenosine Triphosphate / Q000378:metabolism; D002241:Carbohydrates / Q000737:chemistry*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D005958:Glucosephosphates / Q000737:chemistry; D009702:Nucleoside Diphosphate Sugars / Q000096:biosynthesis; D009711:Nucleotides / Q000737:chemistry; D009713:Nucleotidyltransferases / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D010710:Phosphates / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000032:analysis; D014532:Uridine Diphosphate Glucose / Q000737:chemistry; D014544:Uridine Triphosphate / Q000378:metabolism; D015003:Yeasts / Q000201:enzymology / Q000378:metabolism
D002241:Carbohydrates; D004791:Enzyme Inhibitors; D005958:Glucosephosphates; D009702:Nucleoside Diphosphate Sugars; D009711:Nucleotides; D010710:Phosphates; D000255:Adenosine Triphosphate; C031590:glucose-1-phosphate; D009713:Nucleotidyltransferases; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase; D014544:Uridine Triphosphate; D014532:Uridine Diphosphate Glucose