Rate-limiting steps of a stereochemistry retaining β-d-xylosidase from Geobacillus stearothermophilus acting on four substrates.
Kinetic experiments of GSXynB2, a GH52 retaining β-xylosidase, acting on 2-nitrophenyl-β-d-xylopyranoside (2NPX), 4-nitrophenyl-β-d-xylopyranoside (4NPX), 4-methylumbelliferyl-β-d-xylopyranoside (MuX) and xylobiose (X2) were conducted at pH 7.0 and 25 °C. Catalysis proceeds in two steps (xylodidation followed by dexylosidation): E + substrate TO E-xylose + leaving group TO E + xylose. kcat falls into two groups: 4NPX (1.95 s(-1)) and 2NPX, MuX and X2 (15.8 s(-1), 12.6 s(-1), 12.8 s(-1), respectively). Dexylosylation (E-xylose to E + xylose), the common step for the enzymatic hydrolysis of the four substrates, must exceed 15.8 s(-1). kcat of 4NPX would seem mainly limited by xylosylation (step 1) and the other three substrates would seem mainly limited by dexylosylation (step 2) - a conclusion that critically lacks chemical justification (compare 4NPX and 2NPX). Presteady-state rates indicate rapid xylosidation rates for all substrates so a later step (not dexylosidation) is rate-limiting for 4NPX. That 2NPX is an onlier and 4NPX is an outlier (both leaving group pKa of 7.2) of the Brønsted plot pattern (logkcat vs pKa of phenol leaving group) is thus possibly explained by 4NP release. The pH dependency of kcat 2NPX encompasses 2 bell-shaped curves with peaks of pH 3 and pH 7.
USDA-ARS-National Center for Agricultural Utilization Research, Peoria, IL 61604, USA. Electronic address: douglas.jordan@ars.usda.gov.
Douglas B
DB
Jordan
USDA-ARS-National Center for Agricultural Utilization Research, Peoria, IL 61604, USA.
Jay D
JD
Braker
D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013237:Stereoisomerism; D013379:Substrate Specificity; D014995:Xylosidases / Q000378:metabolism*
D014995:Xylosidases
Burst-steady state; Dexylosylation; Glycoside hydrolase; Kinetic data reinterpretation; Rate limitation; β-xylosidase