Garrard L J, Bui Q T, Nygaard R, Raushel F M
J Biol Chem. 1985 May 10;260(9):5548-53.
The pH variation of the kinetic parameters, Vmax and V/K, was examined for the forward and reverse reaction of bovine liver argininosuccinate lyase. In the forward reaction the Vmax profile showed one group that must be unprotonated for activity over the pH range 5-10. The V/K profile for argininosuccinate showed one group that must be unprotonated and two groups that must be protonated for activity. The Vmax profile for the reverse reaction showed only one group that must be protonated for activity. These results support the proposal that catalysis is facilitated in the forward reaction by a general base that abstracts a proton from C-3 of argininosuccinate and a general acid that donates a proton to the guanidinium nitrogen during carbon-nitrogen bond cleavage. The enzyme is completely inactivated by diethyl pyrocarbonate or a water-soluble carbodiimide at pH 6. These experiments suggest that a histidine and a carboxyl group are at or near the active site and are essential for catalytic activity. The observed shifts of the pH profiles of the forward reaction with temperature and organic solvent (25% dioxane) were also consistent with a histidine and carboxylate group.
对牛肝精氨琥珀酸裂解酶的正向和反向反应，研究了动力学参数Vmax和V/K随pH值的变化情况。在正向反应中，Vmax曲线显示在pH值5 - 10范围内有一组必须处于未质子化状态才有活性。精氨琥珀酸的V/K曲线显示有一组必须处于未质子化状态以及两组必须处于质子化状态才有活性。反向反应的Vmax曲线仅显示有一组必须处于质子化状态才有活性。这些结果支持了这样的提议，即在正向反应中，催化作用是由一个从精氨琥珀酸的C - 3提取质子的通用碱和一个在碳 - 氮键断裂期间向胍基氮供质子的通用酸促进的。在pH值6时，该酶被焦碳酸二乙酯或水溶性碳二亚胺完全灭活。这些实验表明，一个组氨酸和一个羧基位于活性位点或其附近，并且对催化活性至关重要。观察到的正向反应pH曲线随温度和有机溶剂（25%二氧六环）的变化也与一个组氨酸和羧酸盐基团相符。
