Author
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Jordan, Douglas |
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Submitted to: Gordon Research Conference Proceedings
Publication Type: Abstract Only Publication Acceptance Date: 8/3/2007 Publication Date: N/A Citation: N/A Interpretive Summary: Technical Abstract: Beta-D-xylosidase from Selenomonas ruminantium is the best catalyst known (kcat, kcat/Km) for promoting hydrolysis of 1,4-beta-D-xylooligosaccharides. **1H NMR experiments indicate the family 43 glycoside hydrolase acts through an inversion mechanism on substrates 4-nitrophenyl-beta-D-xylopyranoside (4NPX) and 1,4-beta-D-xylobiose (X2). Progress curves of 4-nitrophenyl-beta-D-xylobioside, xylotetraose, and xylohexaose reactions indicate that one residue from the nonreducing end of substrate is cleaved per catalytic cycle without processivity. Values of kcat and kcat/Km decrease for xylooligosaccharides longer than X2, illustrating the importance to catalysis of subsites -1 and +1 and the lack there of subsite +2. Homology models of the enzyme active site with docked substrates show that subsites beyond -1 are blocked by protein and subsites beyond +1 are not formed; they suggest D14 and E186 serve catalysis as general base and general acid, respectively. Individual mutations, D14A and E186A, erode kcat and kcat/Km by > 10**3 and to a similar extent for substrates 4NPX and 4-nitrophenyl-alpha-L-arabinofuranoside (4NPA), indicating that the two substrates share the same active site. With 4NPX and 4NPA, pH governs kcat/Km with pKa values of 5 and 7, assigned to D14 and E186, respectively. kcat(4NPX) has a pKa value of 7 and kcat(4NPA) is pH independent above pH 4, suggesting that the catalytically-inactive, “dianionic” enzyme form (D14**–E187**–) binds 4NPX but not 4NPA. |
