title: Mass spectrometry-guided refinement of chemical energy buffers.
abstract: Biocatalytic reactions often require supplying chemical energy and phosphate groups in the form of adenosine triphosphate (ATP). Auxiliary enzymes can be used to convert a reaction by-product-adenosine diphosphate (ADP)-back to ATP. By employing real-time mass spectrometry (RTMS), one can gain an insight into inter-conversions of reactants in multi-enzyme reaction systems and optimize the reaction conditions. In this study, temporal traces of ions corresponding to adenosine monophosphate (AMP), ADP and ATP provided vital information that could be used to adjust activities of the 'buffering enzymes'. Using the RTMS results as a feedback, we also characterized a bienzymatic energy buffer that enables the recovery of ATP in the cases where it is directly hydrolysed to AMP in the main enzymatic reaction. The significance of careful selection of enzyme activities-guided by RTMS-is exemplified in the synthesis of glucose-6-phosphate by hexokinase in the presence of a buffering enzyme, pyruvate kinase. Relative activities of the two enzymes, present in the reaction mixture, influence biosynthetic reaction yields. This observation supports the conclusion that optimization of chemical energy recycling procedures is critical for the biosynthetic reaction economy.
authors.affiliation: Department of Applied Chemistry , National Chiao Tung University , 1001 University Road, Hsinchu 300, Taiwan.
authors.forename: T-R
authors.initials: TR
authors.lastname: Chen
authors.affiliation: Department of Applied Chemistry, National Chiao Tung University, 1001 University Road, Hsinchu 300, Taiwan; Institute of Molecular Science, National Chiao Tung University, 1001 University Road, Hsinchu 300, Taiwan.
authors.forename: P L
authors.identifier: 0000-0003-3471-045X
authors.initials: PL
authors.lastname: Urban
keywords: adenosine triphosphate; biocatalysis; biosynthesis; enzyme; mass spectrometry; nucleotide recycling