Bandivadekar K R, Deshpande V V
Division of Biochemical Sciences, National Chemical Laboratory, Pune, India.
Biochem J. 1996 Apr 15;315 ( Pt 2)(Pt 2):583-7. doi: 10.1042/bj3150583.
Involvement of one out of three tryptophan residues in the active site of the low-molecular-mass xylanase from Chainia has been demonstrated previously [Deshpande, Hinge and Rao (1990) Biochim. Biophys. Acta 1041, 172-177]. The work described here aims at: (i) deducing the structure-function relationship for the tryptophan residue involved at the active site (a) by correlating the effect of N-bromosuccinimide (NBS) on the fluorescence and activity, and (b) by assessing the ability of xylan to protect against decrease in fluorescence versus activity of NBS-treated enzyme; and (ii) probing into the environment of the tryptophan residues by studying the quenching of their fluorescence by various solute quenchers in the presence and absence of guanidine hydrochloride (Gdn.HCl). Complete inactivation of the NBS-treated enzyme occurs well before the loss of fluorescence. Full protection by xylan (0.5%) of the inactivation of enzyme by NBS compared with 30% protection for the decrease in fluorescence confirms the participation of a single tryptophan at the substrate-binding site of the xylanase. The xylanase exhibited a rather low fluorescence emission maximum at 310 nm. There was no shift in the emission maximum on treatment of the enzyme with Gdn.HCl (6.5 M), indicating the rigidity of the microenvironment around tryptophan residues. The quenching studies with acrylamide suggested the occurrence of both collisional as well as static quenching processes. The enzyme retained full activity as well as the characteristic emission maximum at 310 nm in the presence of acrylamide (100 mM), indicating that quenching of fluorescence by acrylamide is a physical process. Acrylamide was more efficient as a quencher than CsCl or KBr. Treatment of the enzyme with Gdn.HCl resulted in an increase in accessibility of the quenchers to the fluorophore as suggested by an increase in the Stern-Volmer quenching constants (K(SV)) of the solute quenchers. The analysis of K(SV) and V values of KBr and CsCl suggests that the overall tryptophan microenvironment in the xylanase from Chainia is slightly electronegative.
先前已证明来自链霉菌的低分子量木聚糖酶活性位点中的三个色氨酸残基之一参与其中[Deshpande、Hinge和Rao（1990年），《生物化学与生物物理学报》1041卷，第172 - 177页]。此处描述的工作旨在：（i）通过关联N - 溴代琥珀酰亚胺（NBS）对荧光和活性的影响，以及评估木聚糖对NBS处理的酶荧光与活性降低的保护能力，推导活性位点所涉及色氨酸残基的结构 - 功能关系；（ii）通过研究在有和没有盐酸胍（Gdn.HCl）存在下各种溶质猝灭剂对色氨酸残基荧光的猝灭，探究色氨酸残基的环境。NBS处理的酶完全失活发生在荧光丧失之前。与荧光降低30%的保护相比，木聚糖（0.5%）对NBS使酶失活具有完全保护作用，这证实了木聚糖酶底物结合位点有单个色氨酸参与。木聚糖酶在310 nm处表现出相当低的荧光发射最大值。用Gdn.HCl（6.5 M）处理酶后，发射最大值没有移动，表明色氨酸残基周围微环境的刚性。用丙烯酰胺进行的猝灭研究表明存在碰撞猝灭和静态猝灭过程。在存在丙烯酰胺（100 mM）的情况下，酶保留了全部活性以及在310 nm处的特征发射最大值，表明丙烯酰胺对荧光的猝灭是一个物理过程。丙烯酰胺作为猝灭剂比CsCl或KBr更有效。用Gdn.HCl处理酶导致溶质猝灭剂的斯特恩 - 沃尔默猝灭常数（K(SV)）增加，这表明猝灭剂对荧光团的可及性增加。对KBr和CsCl的K(SV)和V值分析表明，来自链霉菌的木聚糖酶中色氨酸的整体微环境略带负电。
