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Rate-limiting steps of a stereochemistry retaining β-d-xylosidase from Geobacillus stearothermophilus acting on four substrates
- Douglas B. Jordan, Jay D. Braker
- Archives of biochemistry and biophysics 2015 v.583 pp. 73-78
- Geobacillus stearothermophilus, catalytic activity, enzymatic hydrolysis, glycosidases, pH, phenol, stereochemistry, xylose
- Kinetic experiments of GSXynB2, a ß-xylosidase, acting on 2-nitrophenyl-ß-D-xylopyranoside (2NPX), 4-nitrophenyl-ß-D-xylopyranoside (4NPX), 4-methylumbelliferyl-ß-D-xylopyanoside (MuX) and xylobiose (X2) were conducted at pH 7.0 and 25 °C. Catalysis proceeds in two steps: E + substrate TO E-xylose + leaving group TO E + xylose. k(cat) 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). k(cat) of 4NPX is mainly limited by xylosylation and the other three substrates are mainly limited by dexylosylation. 2NPX is an onlier and 4NPX is an outlier (both leaving group pK(a) of 7.2) of the Brønsted plot pattern (logk(cat) vs pK(a) of phenol leaving group). Burst, steady-state kinetics of 2NPX, MuX and X2 support dexylosylation as rate-limiting. Lack of a burst by 4NPX is consistent with xylosylation being rate-limiting. The pH dependency of k(cat) 2NPX encompasses 2 bell-shaped curves with peaks of pH 3 and pH 7.