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Creative Enzymes presents the most recent development and discoveries in enzyme activity assays. By constantly striving for services of utmost quality, Creative Enzymes has earned the trust of thousands of customers. We are specialized in highly customized and accurate bioanalytical services. Herein, we are glad to provide the most reliable enzymatic assay for pantetheine-phosphate adenylyltransferase.
Coenzyme A (CoA), the principal acyl-group carrier in all living cells, is required for numerous reactions in intermediary metabolism. CoA also plays a critical role in numerous biosynthetic, degradative, and energy-yielding metabolic pathways. In a living cell, CoA is synthesized from pantothenate (vitamin B5), cysteine, and ATP in five steps. Pantetheine-phosphate adenylyltransferase (EC 2.7.7.3; PPAT) catalyzes the penultimate step in this biosynthetic pathway by adenylating 4’-phosphopanthetheine to make dephospho-CoA. Subsequent phosphorylation at the 3’-hydroxyl of the ribose ring by dephospho-CoA kinase (EC 2.7.1.24) produces the acyl-group carrier, CoA. The rate of CoA biosynthesis is regulated by feedback inhibition of the first enzyme of the pathway, pantothenate kinase (EC 2.7.1.33). Studies of the intermediates of CoA biosynthesis have shown that both pantothenate and 4’-phosphopantetheine can accumulate in the cell, suggesting that PPAT may catalyze an additional rate-limiting step in the CoA biosynthesis pathway.
Figure 1: Biosynthesis of CoA in E. Coli.
Reference: Miller J R, et al. Journal of bacteriology, 2007, 189(22): 8196-8205.
PPAT is a member of the nucleotidyltransfer α/β phosphodiesterases family of enzymes, which transfer a nucleotide monophosphate moiety to other substrates. Bacterial PPATs are allosteric homohexamers. PPAT represents an attractive antibacterial target, since it catalyzes a key regulatory step in the CoA biosynthetic pathway and the human PPAT has distinct structures from its bacterial counterparts.
The activity of PPAT can be determined spectrophotometrically by a coupled assay. For instance, PPAT activity is assayed in the reverse direction using hexokinase and glucose-6-phosphate dehydrogenase to couple ATP production to NADP+ reduction. The assay is carried out at room temperature and the change in absorbance at 340 nm is monitored. The quality of our results is assured with the most advanced spectrophotometric instrument. We surpass our competitors with a quick assay turnover in response to the request of either a typical activity determination or a special need. Overall, Creative Enzymes is your trusty-worthy partner and looks forward to win your business as well.
Figure 2: The crystal structure of pantetheine-phosphate adenylyltransferase from E. Coli.
PDB: 1H1T
