Kawaminami S, Ozaki K, Sumitomo N, Hayashi Y, Ito S, Shimada I, Arata Y
Biological Science Laboratories, Kao Corporation, Tochigi, Japan.
J Biol Chem. 1994 Nov 18;269(46):28752-6.
Heteronuclear single-quantum coherence two-dimensional NMR spectroscopy has been used to investigate the active site of endoglucanase K (46 kDa) from Bacillus sp. KSM-330, in which Trp are important for expression of the activity. Endoglucanase K, which was specifically labeled with [indole-2-13C]Trp, was prepared from recombinant Bacillus subtilis that carried the gene for this enzyme on an expression vector, pHSP-KC331. Twelve cross-peaks originating from the C-2 position of Trp residues of endoglucanase K were separately observed in 1H-13C heteronuclear single-quantum coherence spectrum, and six of the cross-peaks have been assigned site-specifically by using site-directed mutagenesis. The chemical shifts of the cross-peaks originating from Trp-174 and Trp-243 were affected by the addition of cellotriose that was used as a competitive inhibitor of the enzyme. On the basis of the NMR data obtained after chemical modification of the enzyme by N-bromosuccinimide, it appears that Trp-174 was oxidized first with retention of 56% of the original activity and Trp-243 was then oxidized with complete loss of activity. Substitution of Trp-174 or Trp-243 by Tyr residue caused a decrease in the specific activity of the enzyme to 49 or 8% of that of the wild-type enzyme, respectively. Km values of these mutant enzymes for p-nitrophenyl beta-D-cellotrioside increased to 5 and 8 times those of the wild-type enzyme, respectively, while kcat values of both of the mutant enzymes decreased to one-fifth of those of the wild-type enzymes. These results suggest that Trp-174 and Trp-243 play an important role in binding of the substrate and/or in the catalytic activity.
异核单量子相干二维核磁共振光谱已被用于研究芽孢杆菌属KSM - 330来源的内切葡聚糖酶K（46 kDa）的活性位点，其中色氨酸对该酶活性的表达很重要。用[吲哚 - 2 - ¹³C]色氨酸特异性标记的内切葡聚糖酶K，是由在表达载体pHSP - KC331上携带该酶基因的重组枯草芽孢杆菌制备的。在¹H - ¹³C异核单量子相干谱中分别观察到12个源自内切葡聚糖酶K色氨酸残基C - 2位置的交叉峰，其中6个交叉峰已通过定点诱变进行了位点特异性归属。源自色氨酸 - 174和色氨酸 - 243的交叉峰的化学位移受纤维三糖添加的影响，纤维三糖用作该酶的竞争性抑制剂。根据用N - 溴代琥珀酰亚胺对该酶进行化学修饰后获得的核磁共振数据，似乎色氨酸 - 174首先被氧化，保留了56%的原始活性，然后色氨酸 - 243被氧化，活性完全丧失。用酪氨酸残基取代色氨酸 - 174或色氨酸 - 243导致该酶的比活性分别降至野生型酶的49%或8%。这些突变酶对对硝基苯基β - D - 纤维三糖苷的Km值分别增加到野生型酶的5倍和8倍，而两种突变酶的kcat值均降至野生型酶的五分之一。这些结果表明色氨酸 - 174和色氨酸 - 243在底物结合和/或催化活性中起重要作用。
