Determination of steady-state kinetic parameters for a xylanase-catalyzed hydrolysis of neutral underivatized xylooligosaccharides by mass spectrometry

A direct mass spectrometric approach was used for the determination of steady-state kinetic parameters, the turnover number (kcat), the Michaelis constant (KM), and the specificity constant (kcat/KM) for an enzyme-catalyzed hydrolysis of xylooligosaccharides. Electrospray ionization mass spectrometry was performed to observe product distributions and to determine kcat, KM, and kcat/KM values for Trichoderma reesei endo-1,4-β-xylanase II (TRX II) with xylohexaose (Xyl6), xylopentaose (Xyl5), xylotetraose (Xyl4), and xylotriose (Xyl3) as substrates. The determined kcat/KM values (0.93, 0.37, 0.027, and 0.00015 μM−1 s−1, respectively) indicated that Xyl6 was the most preferred substrate of TRX II. In addition, the obtained KM value for Xyl5 (136 μM) was roughly twice as high as that for Xyl6 (73 μM), suggesting that at least six putative subsites contribute to the substrate binding in the active site of TRX II. Previous mass spectrometric assays for enzyme kinetics have been used mostly in the case of reactions that result in a transfer of acidic groups (e.g., phosphate) into neutral oligosaccharides giving rise to negatively charged products. Here we demonstrate that such analysis is also feasible in the case of neutral underivatized oligosaccharides. Implications of the results for the catalytic mechanism of TRX II in particular are discussed.