Häberle W, Gruler H, Dutkowski P, Müller-Enoch D
Abteilung Biophysik, Universität Ulm, Bundesrepublik Deutschland.
Z Naturforsch C J Biosci. 1990 Mar-Apr;45(3-4):273-9. doi: 10.1515/znc-1990-3-422.
The light-induced enhancement of the 7-ethoxycoumarin-O-deethylase activity was measured in a reconstituted system, consisting of the enzyme P-450PB-B and the NADPH-cytochrome P-450 reductase. The relative increase of the activity was about 15%. It is shown that the product release process is accelerated by light. The phases of the catalytic cycle of 2 x 10(12) protein complexes were locked by periodic application of light pulses (0.1 s duration, 1.32 s repetition time, and 390-470 nm, 0.27 joule/nmol P-450). More than 80% of the active reconstituted enzyme complexes (= "molecular machines") worked in phase after a few light pulses. The phase relation continued even after switching off the light pulses. The catalytic cycle time was 1.54 s, giving a turnover number of 39 min-1. The turnover number, as determined from the enzyme activity under optimum conditions, was 39 min-1. Due to the dissociation constant of the P-450PB-B:NADPH-P-450 reductase complexes [3] only 24% of the proteins were in the active (= working) state under the conditions used. The lifetime of this complexes is larger than 6 s since more than 4 cycles of the free running enzyme can be observed. This is the first report, that all catalytic active complexes in the test tube can be synchronized by an external light source, if the right repetition time of the pulses is chosen, so that all these "molecular machines" work in phase.
在一个由P-450PB-B酶和NADPH-细胞色素P-450还原酶组成的重组系统中，测定了光诱导的7-乙氧基香豆素-O-脱乙基酶活性的增强。活性的相对增加约为15%。结果表明，光可加速产物释放过程。通过周期性施加光脉冲（持续时间0.1秒，重复时间1.32秒，波长390 - 470纳米，能量0.27焦耳/纳摩尔P-450），锁定了2×10¹²个蛋白质复合物催化循环的各个阶段。经过几次光脉冲后，超过80%的活性重组酶复合物（即“分子机器”）同步工作。即使在光脉冲关闭后，相位关系仍持续存在。催化循环时间为1.54秒，周转数为39分钟⁻¹。在最佳条件下由酶活性测定的周转数为39分钟⁻¹。由于P-450PB-B:NADPH-P-450还原酶复合物的解离常数[3]，在所使用的条件下只有24%的蛋白质处于活性（即工作）状态。这些复合物的寿命大于6秒，因为可以观察到自由运行的酶有超过4个循环。这是第一份报告，即如果选择合适的脉冲重复时间，试管中的所有催化活性复合物可以通过外部光源同步，从而使所有这些“分子机器”同步工作。
