Ordman A B, Kirkwood S
Biochim Biophys Acta. 1977 Mar 15;481(1):25-32. doi: 10.1016/0005-2744(77)90133-4.
Initial velocity and product inhibition studies were carried out on UDP-glucose dehydrogenase (UDPglucose: NAD+ 6-oxidoreductase, EC 1.1.1.22) from beef liver to determine if the kinetics of the reaction are compatible with the established mechanism. An intersecting initial velocity pattern was observed with NAD+ as the variable substrate and UDPG as the changing fixed substrate. UDPglucuronic acid gave competitive inhibition of UDPG and non-competitive inhibition of NAD+. Inhibition by NADH gave complex patterns.Lineweaver-Burk plots of 1/upsilon versus 1/NAD+ at varied levels of NADH gave highly non-linear curves. At levels of NAD+ below 0.05 mM, non-competitive inhibition patterns were observed giving parabolic curves. Extrapolation to saturation with NAD+ showed NADH gave linear uncompetitive inhibition of UDPG if NAD+ was saturating. However, at levels of NAD+ above 0.10 mM, NADH became a competitive inhibitor of NAD+ (parabolic curves) and when NAD+ was saturating NADH gave no inhibition of UDPG. NADH was non-competitive versus UDPG when NAD+ was not saturating. These results are compatible with a mechanism in which UDPG binds first, followed by NAD+, which is reduced and released. A second mol of NAD+ is then bound, reduced, and released. The irreversible step in the reaction must occur after the release of the second mol of NADH but before the release of UDPglucuronic acid. This is apparently caused by the hydrolysis of a thiol ester between UDPglucoronic acid and the essential thiol group of the enzyme. Examination of rate equations indicated that this hydrolysis is the rate-limiting step in the overall reaction. The discontinuity in the velocities observed at high NAD+ concentrations is apparently caused by the binding of NAD+ in the active site after the release of the second mol of NADH, eliminating the NADH inhibition when NAD+ becomes saturating.
对牛肝中的UDP - 葡萄糖脱氢酶（UDP葡萄糖：NAD⁺ 6 - 氧化还原酶，EC 1.1.1.22）进行了初始速度和产物抑制研究，以确定该反应的动力学是否与既定机制相符。以NAD⁺作为可变底物、UDPG作为变化的固定底物时，观察到了相交的初始速度模式。UDP葡萄糖醛酸对UDPG产生竞争性抑制，对NAD⁺产生非竞争性抑制。NADH的抑制作用呈现复杂模式。在不同NADH水平下，1/υ对1/NAD⁺的Lineweaver - Burk图给出高度非线性曲线。在NAD⁺浓度低于0.05 mM时，观察到非竞争性抑制模式，给出抛物线曲线。用NAD⁺外推至饱和表明，如果NAD⁺饱和，NADH对UDPG产生线性非竞争性抑制。然而，在NAD⁺浓度高于0.10 mM时，NADH成为NAD⁺的竞争性抑制剂（抛物线曲线），当NAD⁺饱和时，NADH对UDPG无抑制作用。当NAD⁺不饱和时，NADH对UDPG是非竞争性的。这些结果与一种机制相符，即UDPG首先结合，随后是NAD⁺，NAD⁺被还原并释放。然后结合第二摩尔NAD⁺，还原并释放。反应中的不可逆步骤必须在第二摩尔NADH释放后但在UDP葡萄糖醛酸释放前发生。这显然是由UDP葡萄糖醛酸与酶的必需巯基之间的硫酯水解引起的。速率方程的研究表明，这种水解是整个反应中的限速步骤。在高NAD⁺浓度下观察到的速度不连续性显然是由第二摩尔NADH释放后NAD⁺在活性位点的结合引起的，当NAD⁺饱和时消除了NADH抑制。
