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 (k(cat)), the Michaelis constant (K(M)), and the specificity constant (k(cat)/K(M)) for an enzyme-catalyzed hydrolysis of xylooligosaccharides. Electrospray ionization mass spectrometry was performed to observe product distributions and to determine k(cat), K(M), and k(cat)/K(M) values for Trichoderma reesei endo-1,4-beta-xylanase II (TRX II) with xylohexaose (Xyl(6)), xylopentaose (Xyl(5)), xylotetraose (Xyl(4)), and xylotriose (Xyl(3)) as substrates. The determined k(cat)/K(M) values (0.93, 0.37, 0.027, and 0.00015 microM(-1) s(-1), respectively) indicated that Xyl(6) was the most preferred substrate of TRX II. In addition, the obtained K(M) value for Xyl(5) (136 microM) was roughly twice as high as that for Xyl(6) (73 microM), 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.
Jänis
Janne
J
Department of Chemistry, University of Joensuu, FI-80101 Joensuu, Finland. janne.janis@joensuu.fi
Pulkkinen
Petri
P
Rouvinen
Juha
J
Vainiotalo
Pirjo
P
D002236:Carbohydrate Conformation; D002384:Catalysis; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D013058:Mass Spectrometry; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*
D009844:Oligosaccharides; D043364:Endo-1,4-beta Xylanases