Jayaram H N, Cooney D A, Huang C Y
Laboratory of Pharmacology and Experimental Therapeutics, National Cancer Institute, Bethesda, Maryland 20892.
J Enzyme Inhib. 1986;1(2):151-61. doi: 10.3109/14756368609020113.
Experiments using equilibrium dialysis and fluorescence quenching provided direct evidence that approximately four moles of L-aspartic acid were bound per mole of tetrameric L-asparaginase from Escherichia coli, with a dissociation constant on the order of 60-160 microM. In addition, a set of weaker binding sites with a dissociation constant in the millimolar range were detected. Kinetic studies also revealed that L-aspartic acid inhibited L-asparaginase competitively, with an inhibition constant of 80 microM at micromolar concentrations of L-asparagine; at millimolar concentrations of the amide, an increase in maximal velocity but a decrease in affinity for L-asparagine were observed. L-Aspartic acid at millimolar levels again displayed competitive inhibition. These and other observations suggest that L-aspartic acid binds not only to the active site but also a second site with lower intrinsic affinity for it. The observed "substrate activation" is most likely attributable to the binding of a second molecule of L-asparagine rather than negative cooperativity among the tight sites of the subunits of this tetrameric enzyme. Further support for L-aspartic acid binding to the active site comes from experiments in which the enzyme, when exposed to various group-specific reagents suffered parallel loss of catalytic activity and in its ability to bind L-aspartic acid. Different commercial preparations of Escherichia coli L-asparaginase were found to contain approximately 2-4 moles of L-aspartic acid; these were incompletely removed by dialysis, but could be removed by transamination or decarboxylation. Efficiency of dialysis increased with increasing pH. Taken together, this set of results is consistent with the existence of a covalent beta-aspartyl enzyme intermediate.
使用平衡透析和荧光猝灭的实验提供了直接证据，即每摩尔来自大肠杆菌的四聚体L-天冬酰胺酶结合约四摩尔L-天冬氨酸，解离常数在60 - 160微摩尔范围内。此外，还检测到一组解离常数在毫摩尔范围内的较弱结合位点。动力学研究还表明，L-天冬氨酸竞争性抑制L-天冬酰胺酶，在微摩尔浓度的L-天冬酰胺下抑制常数为80微摩尔；在毫摩尔浓度的酰胺存在下，观察到最大速度增加但对L-天冬酰胺的亲和力降低。毫摩尔水平的L-天冬氨酸再次表现出竞争性抑制。这些及其他观察结果表明，L-天冬氨酸不仅与活性位点结合，还与对其具有较低固有亲和力的第二个位点结合。观察到的“底物激活”最可能归因于第二个L-天冬酰胺分子的结合，而不是这种四聚体酶亚基紧密位点之间的负协同作用。L-天冬氨酸与活性位点结合的进一步证据来自以下实验：当酶暴露于各种基团特异性试剂时，其催化活性和结合L-天冬氨酸的能力同时丧失。发现不同商业制剂的大肠杆菌L-天冬酰胺酶含有约2 - 4摩尔L-天冬氨酸；这些不能通过透析完全去除，但可通过转氨作用或脱羧作用去除。透析效率随pH升高而增加。综上所述，这组结果与共价β-天冬氨酰酶中间体的存在一致。
