Spectroscopic and Kinetic Studies on the Active Site Structure and Substrate Specificities of Pullulanase.

1)在环糊精（CD）与来自肺炎克雷伯氏菌的普鲁兰酶的相互作用中，已知β-CD与α-和γ-CD相比具有最高的亲和力。两条路线的方法已经强烈表明，酶的一些侧链仅包括在β-CD环中。首先，在三种CD中，单甲苯磺酰化β-CD具有最大程度降低的亲和力。2）产气克雷伯氏菌普鲁兰酶的K_m和K_m值与环糊精相比有显著差异<cat>，环糊精对普鲁兰酶的抑制作用也更强。此外，普鲁兰多糖和环糊精引起的差异荧光光谱的形状与肺炎克雷伯氏菌普鲁兰酶的差异荧光光谱的形状有显著差异。3）对产气克雷伯氏菌普鲁兰酶进行了多种定点突变，以确定参与酶反应的重要氨基酸残基的作用。

1) In the interaction of cyclodextrins (CDs) with pullulanase from Klebsiella pneumoniae, it has been known that beta-CD has the highest affinity compared with alpha- and gamma-CDs. Two lines of approach have strongly indicated that some side chain of the enzyme is included in only beta-CD ring. First, mono-tosylated beta-CD among the three CDs has the most decreased affinity. Second, adamantan carboxylate which is known to be included in the ring of beta-CD most remarkably decreased the binding affinity.2) Pullulanase from Klebsiella aerogenes has been found to have significantly different (better) K_m and k_<cat> values and also much stronger inhibition by cyclodextrins. Moreover, the shape of the difference fluorescence spectra caused by pullulan and cyclodextrins are significantly different from those of the enzyme from Klebsiella pneumoniae.3) Several kinds of site-directed mutagenesis of pullulanase from Klebsiella aerogenes were successfully done to determine the roles of amono acid residues importantly involved in the enzyme reaction.