Chemical Modification of the $\beta$-D-Xylosidase from Bacillus stearothermophilus

화학적 수식에 의한 Bacillus stearothermophilus $\beta$-D-Xylosidase 의 연구

  • 서정한 (고려대학교 자연자원대학 유전공학과) ;
  • 최용진 (고려대학교 자연자원대학 유전공학과)
  • Published : 1994.12.01

Abstract

Essential amino acids involving in the catalytic mechanism of the $\beta$-D-xylosidase of Bacillus stearothermophilus were determined by chemical modification studies. Among various che- mical modifiers tested N-bromosuccinimide (NBS), $\rho$-hydroxymercurybenzoate (PHMB), N-ethylma- leimide, 1-[3-(di-ethylamino)-propyl]$-3-ethylcarbodi-imide (EDC), and Woodward's Reagent K(WRK)inactivated the enzyme, resulting in the residual activity of less than 20%. WRK reduced the enzyme activity by modifying carboxylic amino acids, and the inactivation reacion proceeded in the form of pseudo-first-order kinetics. The double-lagarithmic plot of the observed pseudo-first- order rate constant against the modifier concentration yielded a reaction order of 2, indicating that two carboxylic amino acids were essential for the enzyme activity. The $\beta$-D-xylosidase was also inactivated by N-ethylmaleimide which specifically modified a cysteine residue with a reaction order of 1, implying that one cysteine residue was important for the enzyme activity. Xylobiose protected the enzyme against inactivation by WRK and N-ethylmaleimide, revealing that carboxylic amino acids and a cysteine residue were present at the substrate-binding site of the enzyme molecule.

Keywords

${\beta}$-D-Xylosidase;Bacillus stearothermophilus;chemiacal modification;WRK;N-ethylmaleimide;carboxylic amino acids;cysteine

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