Author
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Jordan, Douglas |
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BRAKER, JAY - Former ARS Employee |
Submitted to: Archives of Biochemistry and Biophysics
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 8/10/2015 Publication Date: 10/15/2015 Publication URL: http://handle.nal.usda.gov/10113/62515 Citation: Jordan, D.B., Braker, J.D. 2015. Rate-limiting steps of stereochemistry retaining ß-d-xylosidase from Geobacillus stearothermophilus acting on four substrates. Archives of Biochemistry and Biophysics. 583:73-78. doi: 10.1016/j.abb.2015.08.004. Interpretive Summary: More efficient enzymes for breaking down cellulose and hemicellulose are needed to bring down production costs of bioethanol and other biofuels. This paper corrects some literature mistakes on an enzyme that is used to break down xylan, a major hemicellulose. Its publication will help other researchers in the field avoid the mistakes and be aware of the mistakes as they occur in the literature. In addition, the corrected data itself shows new properties that were not realized before. Technical Abstract: 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. 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 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 pKa of 7.2) of the Brønsted plot pattern (kcat vs pKa 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 kcat 2NPX encompasses 2 bell-shaped curves with peaks of pH 3 and pH 7. |