假单孢菌碱性木聚糖酶分子活性部位的研究

应用多种化学修饰剂对假单孢菌G6－2产生的碱性木聚糖酶A（XynA)进行了修饰。结果表明，作用于色氨酸和谷氨酸（和／或天冬氨酸（的试剂NBS（N－Bromosuccinimide)和WRK（N－Ethyl-5-phenylisoxazolium-3-sulfonate)可分别使酶活明显降低，百作用与丝氨酸，精氨酸，酪氨酸的试剂对酶活无明显影响。XynA的化学修饰动力学分析表明，每个酶分子中有1个色氨酸残基和1个谷氨酸（或天冬氨酸）残基对酶的活性特别重要。NBS的滴定结果表明，木聚糖的加入使可使1个色氨酸残基被保护，木聚糖对NBS的荧光猝来有22％的保护作用。0．2％的燕麦木聚糖可可阻止NBS树XynA的修饰作用；而即使2％的木聚糖也不能阻止WRK的灭活作用，NBS可使XynA的Km增大4倍，而RK可使其Vmax降低三分之二。这些结果表明，色氨酸和谷氨酸（或天冬氨酸（残基位于XynA的活性部位，且前者位于XynA的底物结合中心，后者位于酶的催化中心；图6表1参14

ACTIVE SITES OF ALKALINE XYLANASE MOLECULES

Many modifiers were used to react with alkaline xylanase (XynA). XynA could be inactivated by NBS(N bromosuccinimide) and WRK(N ethyl 5 pheylisoxazolium 3 sulfonate). The kinetic analysis indicated that one tryptophan and one carboxyl group in one molecular of XynA were essential for the XynA activity. Full protection by xylan (0.2%) against the inactivation of enzyme by NBS, compared with 22% protection against the decrease in fluorescence, confirmed that a single tryptophan was at the substrate binding site of the XynA. Xylan had no effect on the modification by WRK. The enzyme modified by NBS showed a little lower V max and the K m was 4 times higher than native XynA, and the enzyme modified by WRK had a similar K m and the V max was one third of that of native XynA. These results revealed that tryptophan was in the substrate binding site and carboxyl in the catalytic site. Fig 6, Tab 1, Ref 14