The role of Thr268 and Phe393 in cytochrome P450BM3
The role of Thr268 and Phe393 in cytochrome P450BM3
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DOI：
10.1016/j.jinorgbio.2005.11.020
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发表时间：
2006-05-01
影响因子：
3.9
通讯作者：
Chapman, Stephen K.
中科院分区：
文献类型：
--
作者：
Clark, Jonathan P.;Miles, Caroline S.;Chapman, Stephen K.
在黄细胞色素P450 BM3中，有几个活性部位残基在整个P450超家族中高度保守。其中，一种苯丙氨酸(Phe393)已被证明通过与隐含保守的血红素配体半胱氨酸相互作用来调节血红素还原潜力。此外，远端苏氨酸(Thr268)参与了多种作用，包括质子捐赠、氧激活和底物识别。P450 BM3中的底物结合导致自旋态从低自旋到高自旋的转变。这种自旋态的变化伴随着还原电势的正移(Delta E-m[WT+花生四烯酸(120µM)]=+138 mV)。Thr268被丙氨酸或天冬酰胺残基取代导致酶通过底物结合产生高自旋复合体的能力显著降低，从而导致底物诱导的电位漂移降低(Delta E-m[T268A+花生四烯酸(120亩M)]=+73 mV，Delta E-m[T268N+花生四烯酸(120亩M)]=+9 mV)。用稳态前停流动力学方法测量了第一电子转移和氧化亚铁衰变的速率常数，发现它们几乎完全依赖于血红素的还原电位。更多的正还原电位导致血红素还原的速率常数增加，含氧亚铁物种更稳定。此外，苏氨酸的取代导致过氧化氢的产量增加，而不是羟化产物。这些结果表明，这个活性部位苏氨酸在底物识别和维持有效功能的酶中起着重要作用。然而，氧化亚铁衰变的速率常数与还原电位的依赖关系提出了一些问题，即Thr268在铁氧稳定中的重要性。(C)2005 Elsevier Inc.保留所有权利。
in flavocytochrome P450 BM3 there are several active site residues that are highly conserved throughout the P450 superfamily. Of these, a phenylalanine (Phe393) has been shown to modulate heme reduction potential through interactions with the implicitly conserved heme-ligand cysteine. In addition, a distal threonine (Thr268) has been implicated in a variety of roles including proton donation, oxygen activation and substrate recognition. Substrate binding in P450 BM3 causes a shift in the spin state from low- to high-spin. This change in spin-state is accompanied by a positive shift in the reduction potential (Delta E-m [WT + arachidonate (120 mu M)] = +138 mV). Substitution of Thr268 by an alanine or asparagine residue causes a significant decrease in the ability of the enzyme to generate the high-spin complex via substrate binding and consequently leads to a decrease in the substrate-induced potential shift (Delta E-m [T268A + arachidonate (120 mu M)] = +73 mV, Delta E-m [T268N + arachidonate (120 mu M)] = +9 mV). Rate constants for the first electron transfer and for oxy-ferrous decay were measured by pre-steady-state stopped-flow kinetics and found to be almost entirely dependant on the heme reduction potential. More positive reduction potentials lead to enhanced rate constants for heme reduction and more stable oxy-ferrous species. In addition, substitutions of the threonine lead to an increase in the production of hydrogen peroxide in preference to hydroxylated product. These results suggest an important role for this active site threonine in substrate recognition and in maintaining an efficiently functioning enzyme. However, the dependence of the rate constants for oxy-ferrous decay on reduction potential raises some questions as to the importance of Thr268 in iron-oxo stabilisation. (c) 2005 Elsevier Inc. All rights reserved.