Effects of isoleucine 135 side chain length on the cofactor donor-acceptor distance within F420H2:NADP+ oxidoreductase: A kinetic analysis.
Effects of isoleucine 135 side chain length on the cofactor donor-acceptor distance within F420H2:NADP+ oxidoreductase: A kinetic analysis.
复制标题
DOI：
10.1016/j.bbrep.2016.11.012
复制
发表时间：
2017-03-01
影响因子：
2.7
通讯作者：
Johnson-Winters, Kayunta
中科院分区：
文献类型：
--
作者：
Le, Cuong Quang;Oyugi, Mercy;Johnson-Winters, Kayunta
F420H2：NADP+氧化还原酶(FNO)通过从还原的F420辅因子转移氢化物，催化NADP+可逆还原为NADPH。在这里，我们采用了wtFNO和异亮氨酸135(I135)FNO变体的结合研究、稳态和稳态前动力学方法来研究侧链长度对NADP+和F420前体FO之间供体-受体距离的影响。FNO保守的I135残基被转化为缬氨酸、丙氨酸和甘氨酸，从而缩短了侧链长度。WtFno及其变异体的稳态动力学分析显示，随FO浓度的变化符合经典的Michaelis-Menten动力学。数据显示，随着侧链长度的减少，随着FO浓度的变化，kcat降低。稳态曲线显示了NADPH变化时的非米氏动力学行为。NADPH浓度变化的双倒数曲线呈向下凹形，而NADPH结合曲线的Hill系数小于1。这些数据表明两个相同的单体之间存在负的协同作用。稳态前的Abs420-时间轨迹表现为两相动力学，有快相(氢化物转移)和慢相。快相的速率常数随侧链长度的减小而增大。第二阶段的速率常数对于每个变体保持~2s-1。我们的数据表明，I135在维持两个辅助因子之间的供体-受体距离方面发挥了关键作用，从而调节了氢化物从FOH2转移到NADP+的速度。因此，FNO是一种动态调节NADPH产生的酶。
F420H2:NADP+ Oxidoreductase (Fno) catalyzes the reversible reduction of NADP+ to NADPH by transferring a hydride from the reduced F420 cofactor. Here, we have employed binding studies, steady-state and pre steady-state kinetic methods upon wtFno and isoleucine 135 (I135) Fno variants in order to study the effects of side chain length on the donor-acceptor distance between NADP+ and the F420 precursor, FO. The conserved I135 residue of Fno was converted to a valine, alanine and glycine, thereby shortening the side chain length. The steady-state kinetic analysis of wtFno and the variants showed classic Michaelis-Menten kinetics with varying FO concentrations. The data revealed a decreased kcat as side chain length decreased, with varying FO concentrations. The steady-state plots revealed non-Michaelis-Menten kinetic behavior when NADPH was varied. The double reciprocal plot of the varying NADPH concentrations displays a downward concave shape, while the NADPH binding curves gave Hill coefficients of less than 1. These data suggest that negative cooperativity occurs between the two identical monomers. The pre steady-state Abs420 versus time trace revealed biphasic kinetics, with a fast phase (hydride transfer) and a slow phase. The fast phase displayed an increased rate constant as side chain length decreased. The rate constant for the second phase, remained ~2s-1 for each variant. Our data suggest that I135 plays a key role in sustaining the donor-acceptor distance between the two cofactors, thereby regulating the rate at which the hydride is transferred from FOH2 to NADP+. Therefore, Fno is a dynamic enzyme that regulates NADPH production.