Kinetic analysis of NodST sulfotransferase using an electrospray ionization mass spectrometry assay.
A novel and efficient enzyme kinetics assay using electrospray ionization mass spectrometry was developed and applied to the bacterial carbohydrate sulfotransferase (NodST). NodST catalyzes the sulfuryl group transfer from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to chitobiose, generating 3'-phosphoadenosine 5'-phosphate (PAP) and chitobiose-6-OSO(3)(-) as products. Traditional spectrophotometric assays are not applicable to the NodST system since no shift in absorption accompanies sulfuryl group transfer. Alternative assays have employed thin-layer chromatography, but this procedure is time-consuming and requires radioactive materials. The ESI-MS assay presented herein requires no chromophoric substrate or product, and the analysis time is very short. The ESI-MS assay is used to determine NodST kinetic parameters, including K(M), V(max), and K(i) (for PAP). In addition, the mode of inhibition for PAP was rapidly determined. The results were in excellent agreement with those obtained from previous assays, verifying the accuracy and reliability of the ESI-MS assay. This unique technique is currently being used to investigate the enzymatic mechanism of NodST and to identify sulfotransferase inhibitors.
Pi
Na
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Department of Chemistry, University of California, Berkeley, California 94720, USA.
Armstrong
Joshua I
JI
Bertozzi
Carolyn R
CR
Leary
Julie A
JA
D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry*; D002384:Catalysis; D004187:Disaccharides / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D010724:Phosphoadenosine Phosphosulfate / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D000097811:Carbohydrate Sulfotransferases
D001426:Bacterial Proteins; D004187:Disaccharides; D004791:Enzyme Inhibitors; D010724:Phosphoadenosine Phosphosulfate; D015238:Sulfotransferases; D000097811:Carbohydrate Sulfotransferases; C032438:chitobiose; C092976:NodH protein, Sinorhizobium meliloti