Department of Chemistry, University of Georgia, Athens, GA 30602, USA.
Biochemistry. 2012 Aug 21;51(33):6527-33. doi: 10.1021/bi300632k. Epub 2012 Aug 10.
The effects of pH and hydrostatic pressure on the reaction of H463F tryptophan indole-lyase (TIL) have been evaluated. The mutant TIL shows very low activity for elimination of indole but is still competent to form a quinonoid intermediate from l-tryptophan [Phillips, R. S., Johnson, N., and Kamath, A. V. (2002) Biochemistry 41, 4012-4019]. Stopped-flow measurements show that the formation of the quinonoid intermediate at 505 nm is affected by pH, with a bell-shaped dependence for the forward rate constant, k(f), and dependence on a single basic group for the reverse rate constant, k(r), with the following values: pK(a1) = 8.14 ± 0.15, pK(a2) = 7.54 ± 0.15, k(f,min) = 18.1 ± 1.3 s(-1), k(f,max) = 179 ± 46.3 s(-1), k(r,min) = 11.4 ± 1.2 s(-1), and k(r,max) = 33 ± 1.6 s(-1). The pH effects may be due to ionization of Tyr74 as the base and Cys298 as the acid influencing the rate constant for deprotonation. High-pressure stopped-flow measurements were performed at pH 8, which is the optimum for the forward reaction. The rate constants show an increase with pressure up to 100 MPa and a subsequent decrease above 100 MPa. Fitting the pressure data gives the following values: k(f,0) = 15.4 ± 0.8 s(-1), ΔV(‡) = -29.4 ± 2.9 cm(3) mol(-1), and Δβ(‡) = -0.23 ± 0.03 cm(3) mol(-1) MPa(-1) for the forward reaction, and k(r,0) = 20.7 ± 0.8 s(-1), ΔV(‡) = -9.6 ± 2.3 cm(3) mol(-1), and Δβ(‡) = -0.05 ± 0.02 cm(3) mol(-1) MPa(-1) for the reverse reaction. The primary kinetic isotope effect on quinonoid intermediate formation at pH 8 is small (~2) and is not significantly pressure-dependent, suggesting that the effect of pressure on k(f) may be due to perturbation of an active site preorganization step. The negative activation volume is also consistent with preorganization of the ES complex prior to quinonoid intermediate formation, and the negative compressibility may be due to the effect of pressure on the enzyme conformation. These results support the conclusion that the preorganization of the H463F TIL Trp complex, which is probably dominated by motion of the l-Trp indole moiety of the aldimine complex, contributes to quinonoid intermediate formation.
已评估 pH 值和静压对 H463F 色氨酸吲哚-lyase（TIL）反应的影响。该突变体 TIL 对吲哚的消除活性非常低，但仍能从 l-色氨酸形成醌型中间体[Phillips，R.S.，Johnson，N.，和 Kamath，A.V.（2002）生物化学 41，4012-4019]。停流测量显示，505nm 处醌型中间体的形成受 pH 值影响，正向速率常数 k(f)呈钟形依赖性，反向速率常数 k(r)依赖于单个碱性基团，其值如下：pK(a1)=8.14±0.15，pK(a2)=7.54±0.15，k(f,min)=18.1±1.3s(-1)，k(f,max)=179±46.3s(-1)，k(r,min)=11.4±1.2s(-1)，k(r,max)=33±1.6s(-1)。pH 值的影响可能是由于 Tyr74 作为碱和 Cys298 作为酸的离解，影响去质子化的速率常数。在 pH 8 下进行了高压停流测量，这是正向反应的最佳 pH 值。速率常数在 100 MPa 以下随压力增加而增加，在 100 MPa 以上随压力增加而减小。拟合压力数据得到以下值：k(f,0)=15.4±0.8s(-1)，ΔV(‡)=-29.4±2.9cm(3)mol(-1)，Δβ(‡)=-0.23±0.03cm(3)mol(-1)MPa(-1)对于正向反应，k(r,0)=20.7±0.8s(-1)，ΔV(‡)=-9.6±2.3cm(3)mol(-1)，Δβ(‡)=-0.05±0.02cm(3)mol(-1)MPa(-1)对于反向反应。在 pH 8 时形成醌型中间体的初级动力学同位素效应较小（约为 2），且对压力的依赖性不明显，表明压力对 k(f)的影响可能是由于活性位点预组织步骤受到干扰。负的活化体积也与形成醌型中间体之前 ES 复合物的预组织一致，负的可压缩性可能是由于压力对酶构象的影响。这些结果支持这样的结论，即 H463F TIL Trp 复合物的预组织，可能由醛亚胺复合物中 l-Trp 吲哚部分的运动主导，有助于醌型中间体的形成。
