Sloan M J, Phillips R S
Department of Chemistry, University of Georgia, Athens 30602-2556, USA.
Biochemistry. 1996 Dec 17;35(50):16165-73. doi: 10.1021/bi961211c.
Tryptophan indole-lyase catalyzes the hydrolytic cleavage of L-tryptophan to indole and ammonium pyruvate. After the enzyme is mixed with L-tryptophan in the rapid-scanning stopped-flow spectrophotometer, there is an absorbance increase at 505 nm in the pre-steady state attributed to formation of a quinonoid intermediate, which occurs in at least three consecutive first-order phases. Reaction with [alpha-2H]-L-tryptophan results in significant primary kinetic isotope effects on the first two phases, and there is a significant isotope effect on the amplitude of the absorbance increase in the second phase. This result suggests that proton transfer to carbon to form the indolenine intermediate is relatively slow and is probably at least partially rate-determining. Reaction of L-tryptophan in the presence of benzimidazole results in a rapid increase in absorbance in the first phase, followed by a decrease in absorbance in the second phase, with rate constants very similar to those observed without benzimidazole. We have also examined aza and thia analogs of L-tryptophan, with the benzene ring of the indole replaced by pyridine or thiophene. Both 4,5-thiatryptophan and 6,7-thiatryptophan form quinonoid intermediates in the reaction with tryptophan indole-lyase; however, 6,7-thiatryptophan is a better substrate (kcat/K(m) = 32% of L-trp) for tryptophan indole-lyase than is 4,5-thiatryptophan (kcat/K(m) = 4% of L-trp). Benzimidazole affects the pre-steady-state reaction of 6,7-thiatryptophan in a way similar to L-tryptophan, while benzimidazole does not affect the pre-steady-state reaction of 4,5-thiatryptophan. 4-Aza-, 5-aza-, 6-aza-, and 7-aza-L-tryptophan are all very slow substrates (kcat < 1% of L-trp) for Escherichia coli tryptophan indole-lyase. beta-Indazolyl-L-alanine is a relatively good substrate and exhibits a quinonoid intermediate in its reaction with tryptophan indole-lyase. 6-Aza- and 7-azatryptophan accumulate quinonoid intermediates in the reaction with tryptophan indole-lyase, whereas 4-aza- and 5-azatryptophans do not significantly accumulate quinonoid intermediates, and these latter compounds exhibit very high K(m) values. Addition of benzimidazole does not change the rapid-scanning stopped-flow spectra of 6-aza- and 7-azatryptophan. This suggests that the rate-determining step in the reaction changes depending on the position and type of heteroatom substitution. For 6-aza- and 7-azatryptophan, the very slow rates of elimination may be due to slow C-protonation of the azaindole, while for 4,5-thiatryptophan, the elimination of thienopyrrole is probably slow. Of all analogs examined, 6,7-thiatryptophan is most similar to tryptophan in its reaction with E. coli tryptophan indole-lyase.
色氨酸吲哚裂解酶催化L-色氨酸水解裂解为吲哚和丙酮酸铵。在快速扫描停流分光光度计中，该酶与L-色氨酸混合后，在预稳态下505nm处吸光度增加，这归因于醌类中间体的形成，该过程至少发生在三个连续的一级反应阶段。与[α-2H]-L-色氨酸反应对前两个阶段产生显著的一级动力学同位素效应，并且对第二阶段吸光度增加的幅度有显著的同位素效应。这一结果表明质子转移到碳上形成吲哚烯中间体的过程相对较慢，可能至少部分是速率决定步骤。L-色氨酸在苯并咪唑存在下反应，第一阶段吸光度迅速增加，随后第二阶段吸光度下降，其速率常数与无苯并咪唑时观察到的非常相似。我们还研究了L-色氨酸的氮杂和硫杂类似物，吲哚的苯环被吡啶或噻吩取代。4,5-硫代色氨酸和6,7-硫代色氨酸在与色氨酸吲哚裂解酶反应中均形成醌类中间体；然而，对于色氨酸吲哚裂解酶而言，6,7-硫代色氨酸（kcat/K(m)=L-色氨酸的32%）比4,5-硫代色氨酸（kcat/K(m)=L-色氨酸的4%）是更好的底物。苯并咪唑以类似于L-色氨酸的方式影响6,7-硫代色氨酸的预稳态反应，而苯并咪唑不影响4,5-硫代色氨酸的预稳态反应。4-氮杂-L-色氨酸、5-氮杂-L-色氨酸、6-氮杂-L-色氨酸和7-氮杂-L-色氨酸都是大肠杆菌色氨酸吲哚裂解酶的非常缓慢的底物（kcat<L-色氨酸的1%）。β-吲唑基-L-丙氨酸是一种相对较好的底物，在与色氨酸吲哚裂解酶反应中表现出醌类中间体。6-氮杂色氨酸和7-氮杂色氨酸在与色氨酸吲哚裂解酶反应中积累醌类中间体，而4-氮杂色氨酸和5-氮杂色氨酸不会显著积累醌类中间体，并且后两种化合物表现出非常高的K(m)值。添加苯并咪唑不会改变6-氮杂色氨酸和7-氮杂色氨酸的快速扫描停流光谱。这表明反应中的速率决定步骤根据杂原子取代的位置和类型而变化。对于6-氮杂色氨酸和7-氮杂色氨酸，非常缓慢的消除速率可能是由于氮杂吲哚的C-质子化缓慢，而对于4,5-硫代色氨酸，噻吩并吡咯的消除可能很慢。在所有研究的类似物中，6,7-硫代色氨酸在与大肠杆菌色氨酸吲哚裂解酶反应中与色氨酸最为相似。
