Bonagura C A, Sundaramoorthy M, Bhaskar B, Poulos T L
Department of Molecular Biology & Biochemistry, University of California, Irvine 92697-3900, USA.
Biochemistry. 1999 Apr 27;38(17):5538-45. doi: 10.1021/bi982996k.
Earlier work [Bonagura et al. (1996) Biochemistry 35, 6107] showed that the K+ site found in the proximal pocket of ascorbate peroxidase (APX) could be engineered into cytochrome c peroxidase (CCP). Binding of K+ at the engineered site results in a loss in activity and destabilization of the CCP compound I Trp191 cationic radical owing to long-range electrostatic effects. The engineered CCP mutant crystal structure has been refined to 1.5 A using data obtained at cryogenic temperatures which provides a more detailed basis for comparison with the naturally occurring K+ site in APX. The characteristic EPR signal associated with the Trp191 radical becomes progressively weaker as K+ is added, which correlates well with the loss in enzyme activity as [K+] is increased. These results coupled with stopped-flow studies support our earlier conclusions that the loss in activity and EPR signal is due to destabilization of the Trp191 cationic radical.
早期的研究工作[博纳古拉等人（1996年），《生物化学》第35卷，6107页]表明，抗坏血酸过氧化物酶（APX）近端口袋中的钾离子位点可以被引入细胞色素c过氧化物酶（CCP）中。钾离子在工程化位点的结合会导致活性丧失以及CCP复合I色氨酸191阳离子自由基的不稳定，这是由于长程静电效应所致。利用在低温下获得的数据，已将工程化CCP突变体的晶体结构精修至1.5埃，这为与APX中天然存在的钾离子位点进行比较提供了更详细的基础。随着钾离子的加入，与色氨酸191自由基相关的特征性电子顺磁共振（EPR）信号逐渐减弱，这与随着[钾离子]增加酶活性的丧失密切相关。这些结果与停流研究相结合，支持了我们早期的结论，即活性和EPR信号的丧失是由于色氨酸191阳离子自由基的不稳定所致。
