Time-resolved Mass Spectrometry Studies of Enzyme Kinetics

3 Time-resolved Mass Spectrometry Studies of Enzyme Kinetics 

Mass spectrometry can characterize enzyme kinetics, mechanism, and selectivity [27]. Notably, MS enables kinetic analyses of enzymatic reactions where it is not possible to carry out simple spectrophotometric assays - for example, in the absence of chromogenic substrates [28, 29]. 

Michaelis-Menten kinetics is used to describe the progress of enzymatic reactions, which can be summarized by the following simplified equation [30]  

Enzyme reaction intermediates can be characterized, in sub-second timescale, using the so-called pulsed flow method [35]. It employs a direct on-line interface between a rapid-mixing device and a ESI-MS system. It circumvents chemical quenching. By way of this strategy, it was possible to detect the intermediate of a reaction catalyzed by 5-enolpyruvoyl-shikimate-3-phosphate synthase [35]. The time-resolved ESI-MS method was also implemented in measurements of pre-steady-state kinetics of an enzymatic reaction involving Bacillus circulans xylanase [36]. The pre-steady-state kinetic parameters for the formation of the covalent intermediate in the mutant xylanase were determined. The MS results were in agreement with those obtained by stopped-flow ultraviolet-visible spectroscopy. In a later work, hydrolysis of p-nitrophenyl acetate by chymotrypsin was used as a model system [27]. The chymotrypsin-catalyzed hydrolysis follows the mechanism [27]  

Under steady-state conditions, [EP2], [E], and [ES] are approximately constant while [PJ and [P2] increase over time. While the Michaelis-Menten kinetics describes behavior of the steady-state system, using a special reaction chamber with adjustable volume [37] 

---