Trost P, Pupillo P
Department of Biology, University of Bologna, Italy.
Arch Biochem Biophys. 1993 Oct;306(1):76-82. doi: 10.1006/abbi.1993.1483.
D-Glyceraldehyde 3-phosphate: NADP+ oxidoreductase, nonphosphorylating (GNR; EC 1.2.1.9) purified from spinach leaves was investigated by initial velocity analysis. The hyperbolic saturation curves became nonhyperbolic when NADP+ was varied at elevated D-glyceraldehyde 3-phosphate (G3P) concentrations (sigmoidicity) or when G3P was varied at low NADP+ concentrations (pseudo-substrate inhibition), suggesting a random bi bi mechanism (Scagliarini et al. Plant Physiol. 94, 1337-1344, 1990). Free ATP was a linear competitive inhibitor of both NADP+ with KI 0.5 +/- 0.2 mM (SD) and G3P with KI 3.2 +/- 0.2 mM as determined by data in the hyperbolic range of responses when the nonvaried substrate was saturating. Similarly ADP inhibited competitively with KI 1.9 +/- 0.4 mM (NADP+) and 3.5 +/- 0.5 mM (G3P). Inhibition was mixed-type when the nonvaried substrate was below saturation. ATP, but not ADP, tended to enhance the nonhyperbolic behavior of GNR, resulting in potentiated inhibition at high [G3P]/[NADP+] ratios. The Mg-chelated form of ATP was less effective. The rate equation of a steady state random bi bi reaction mechanism in the presence of a dead-end inhibitor was derived. Suitable values of the rate constants were chosen to fit the kinetic data for the uninhibited enzyme. These values and the measured inhibition constants inserted in the rate equation can satisfactorily account for the nonhyperbolic inhibition patterns of ATP and ADP. The generalized model represents a possible alternative to allosteric models in interpreting nonlinear kinetics and dead-end inhibition of two-substrate enzymes.
对从菠菜叶中纯化得到的非磷酸化D-甘油醛-3-磷酸：NADP⁺氧化还原酶（GNR；EC 1.2.1.9）进行了初速度分析。当在较高的D-甘油醛-3-磷酸（G3P）浓度下改变NADP⁺时（S形曲线），或者在低NADP⁺浓度下改变G3P时（假底物抑制），双曲线饱和曲线变成了非双曲线，这表明其为随机双双机制（斯卡利亚里尼等人，《植物生理学》94，1337 - 1344，1990）。游离ATP是NADP⁺的线性竞争性抑制剂，其抑制常数KI为0.5±0.2 mM（标准差），也是G3P的线性竞争性抑制剂，其抑制常数KI为3.2±0.2 mM，这是在非变化底物饱和时，根据双曲线响应范围内的数据测定的。同样，ADP也具有竞争性抑制作用，其抑制常数KI为1.9±0.4 mM（针对NADP⁺）和3.5±0.5 mM（针对G3P）。当非变化底物低于饱和浓度时，抑制作用为混合型。ATP（而非ADP）倾向于增强GNR的非双曲线行为，导致在高[G3P]/[NADP⁺]比值下抑制作用增强。Mg螯合形式的ATP效果较差。推导了存在终产物抑制剂时稳态随机双双反应机制的速率方程。选择了合适的速率常数来拟合未受抑制酶的动力学数据。将这些值以及测得的抑制常数代入速率方程，可以令人满意地解释ATP和ADP的非双曲线抑制模式。在解释双底物酶的非线性动力学和终产物抑制方面，该通用模型是变构模型的一种可能替代方案。
