Wedler F C, Ley B W
Department of Molecular and Cell Biology, Pennsylvania State University, University Park 16802.
Arch Biochem Biophys. 1993 Mar;301(2):416-23. doi: 10.1006/abbi.1993.1165.
Changes in the kinetic properties of homoserine dehydrogenase-I (HD-I) from Escherichia coli, caused by substitution of Na+ for the normal activating monovalent ion, K+, has been investigated by equilibrium isotope exchange kinetics (EIEK). HD-I, part of the aspartokinase/homoserine dehydrogenase-I complex, is one of the few dehydrogenases to exhibit allosteric feedback regulation and cation activation. EIEK methods are especially useful for definitively identifying which rate constants are altered by bound modifiers. Saturation curves for the [14C]Hse<-->ASA and [3H]NADP+<-->NADPH exchanges were compared in the presence of K+ vs Na+, varying different combinations of substrate pairs in constant ratio at equilibrium. Kinetic differences between the K+ and Na+ enzymes were analyzed systematically by simulations with the ISOBI program. This analysis clearly demonstrates that substituting Na+ for K+ shifts the kinetic mechanism from preferred order random to a nearly random order scheme, along with causing significant rate limitation at catalysis between the central complexes. Initial velocity kinetics demonstrate that HD-I has a 10-fold higher affinity for Na+ than K+, but that the Na(+)-enzyme is 10-fold less active and exhibits higher substrate Km values, especially for L-Hse.
通过平衡同位素交换动力学（EIEK）研究了用Na⁺替代正常激活单价离子K⁺导致的大肠杆菌高丝氨酸脱氢酶-I（HD-I）动力学性质的变化。HD-I是天冬氨酸激酶/高丝氨酸脱氢酶-I复合物的一部分，是少数表现出变构反馈调节和阳离子激活的脱氢酶之一。EIEK方法对于明确确定哪些速率常数会因结合的修饰剂而改变特别有用。在存在K⁺和Na⁺的情况下，比较了[¹⁴C]高丝氨酸⇄天冬氨酸半醛和[³H]NADP⁺⇄NADPH交换的饱和曲线，在平衡时以恒定比例改变底物对的不同组合。通过使用ISOBI程序进行模拟，系统地分析了K⁺和Na⁺酶之间的动力学差异。该分析清楚地表明，用Na⁺替代K⁺会使动力学机制从优先顺序随机转变为几乎随机顺序的模式，同时在中心复合物之间的催化过程中造成显著的速率限制。初始速度动力学表明，HD-I对Na⁺的亲和力比对K⁺高10倍，但Na⁺酶的活性低10倍，并且表现出更高的底物Km值，特别是对于L-高丝氨酸。
