Phillips R S, Doshi K J
Department of Chemistry, and Center for Metalloenzyme Studies, University of Georgia, Athens 30602-2556, USA.
Eur J Biochem. 1998 Jul 15;255(2):508-15. doi: 10.1046/j.1432-1327.1998.2550508.x.
Escherichia coli tryptophan indole-lyase (Trpase) is a pyridoxal 5'-phosphate(pyridoxal-P)-dependent enzyme which catalyzes the hydrolytic cleavage of L-tryptophan to indole and ammonium pyruvate. This enzyme is strongly activated by K+ and similar monovalent cations, and the spectrum of the pyridoxal-P cofactor is also affected by pH and cations. Treatment of Trpase with trypsin results in a 20-100-fold decrease in elimination activity, depending on the substrate, concomitant with a change in the relative amounts of the 337 nm and 420 nm forms of the bound pyridoxal-P, and a shift in the lambda(max) from 420 nm to 423 nm. In addition, the pH sensitivity of the pyridoxal-P cofactor is eliminated after trypsin treatment. Nicked Trpase exhibits only fourfold activation by K+, compared with about 50-fold for native enzyme, but the K(A) for K+ is unaffected. Both the native and trypsin-nicked Trpase react with amino acids to form equilibrating mixtures of external aldimine and quinonoid intermediates in rapid-scanning stopped-flow experiments. However, the rate constant for quinonoid intermediate formation from L-tryptophan is reduced by at least 400-fold by treatment with trypsin. In contrast, the rate constant for formation of quinonoid intermediates of L-alanine and S-ethyl-L-cysteine is affected only twofold or less by trypsin treatment. The site of trypsin cleavage was identified by electrospray-ionization mass spectrometry as Lys406, which is predicted to lie on a flexible surface loop. Some active-site residues, particularly Arg419, which is predicted by sequence similarity to be the substrate alpha-carboxylate-binding site, and His463, are located in the sequence between Lys406 and the C-terminus. Hence, cleavage of the peptide bond of E. coli Trpase at Lys406 probably affects the change from active to inactive conformations that normally takes place in the presence of activating monovalent cations.
大肠杆菌色氨酸吲哚裂解酶（色氨酸酶）是一种依赖于磷酸吡哆醛（磷酸吡哆醛）的酶，它催化L-色氨酸水解裂解为吲哚和丙酮酸铵。该酶被K⁺和类似的单价阳离子强烈激活，并且磷酸吡哆醛辅因子的光谱也受pH值和阳离子的影响。用胰蛋白酶处理色氨酸酶会导致消除活性降低20至100倍，这取决于底物，同时结合的磷酸吡哆醛的337nm和420nm形式的相对量会发生变化，并且最大吸收波长从420nm移至423nm。此外，胰蛋白酶处理后，磷酸吡哆醛辅因子的pH敏感性消失。与天然酶约50倍的激活相比，切口色氨酸酶仅被K⁺激活四倍，但K⁺的解离常数不受影响。在快速扫描停流实验中，天然色氨酸酶和胰蛋白酶切口色氨酸酶都与氨基酸反应形成外部醛亚胺和醌类中间体的平衡混合物。然而，用胰蛋白酶处理后，L-色氨酸形成醌类中间体的速率常数降低了至少400倍。相比之下，L-丙氨酸和S-乙基-L-半胱氨酸醌类中间体的形成速率常数仅受胰蛋白酶处理影响两倍或更小。通过电喷雾电离质谱法确定胰蛋白酶切割位点为Lys406，预计该位点位于柔性表面环上。一些活性位点残基，特别是通过序列相似性预测为底物α-羧酸盐结合位点的Arg419和His463，位于Lys406和C末端之间的序列中。因此，大肠杆菌色氨酸酶在Lys406处的肽键断裂可能会影响通常在存在激活单价阳离子的情况下从活性构象到非活性构象的转变。
