A new method to investigate the catalytic mechanism of YhdE pyrophosphatase by using a pyrophosphate fluorescence probe.

摘要: YhdE is a Maf (multicopy associated filamentation) proteins from Escherichia coli which exhibits pyrophosphatase activity towards selected nucleotides, although its catalytic mechanism remains unclear. Herein we used novel fluorescence probe (4-isoACBA–Zn(II) complex) to characterize the enzymatic properties of and mutant, establishing new method for assaying function. Our results reveal first time that sensor confers high sensitivity specificity pyrophosphate (PPi) direct product YhdE. Crystal structures mutant in active-site loop (YhdE_E33A) show conformational flexibility implicated ITC experiments computational docking further Asp70 substrate dTTP coordinate Mn2+. Quantum mechanics calculations indicate hydrolysis appears follow stepwise pathway water molecule attacks α-phosphorus atom substrate, followed by release PPi pentavalent intermediate.