Direct measurement of proton release by yeast enolase upon binding magnesium ions
Direct measurement of proton release by yeast enolase upon binding magnesium ions
复制标题
结合镁离子后酵母烯醇酶直接测量质子释放
DOI：
10.1016/0014-5793(74)81162-2
复制
发表时间：
1974
期刊：
影响因子：
3.5
通讯作者：
A. Johnson
中科院分区：
文献类型：
--
作者：
L. Faller;A. Johnson
我们最近报道了二价金属离子与酵母烯醇化酶（2-磷酸-D-甘油酸水解酶，EC 4.2。1. l 1）耦合到质子平衡[11.这一结论是基于流动微量热测量的热金属结合在三个缓冲液具有不同的电离热。当脱辅基酶[2]上的两个特异性阳离子结合位点在pH 7.5和0.05 M离子强度下被Mg+离子填充时，热数据显示与两个质子的释放一致。量热实验无法区分每个Mg*+离子结合时释放一个质子和响应于第一个Mg*+离子结合引发的构象变化释放两个质子[3，4]。在单一pH下的量热测量也无法确定所涉及的可滴定基团的数量或身份。我们报告直接测量质子释放酵母烯醇化酶结合镁离子进行澄清这些点。质子释放-pH曲线为不对称的钟形曲线。在pH 6.5时释放的质子数最多，超过2个，表明金属结合中有两个以上的可滴定基团。pH 6.5以上的曲线形状与金属与组氨酰残基的络合一致。高于pH 7两个Mg*+离子结合与解离常数G 5 PM，质子释放与金属结合相一致。烯醇化酶对Mg 2+离子的亲和力在低于pH 7时降低。在pH 6.5以下释放的质子数急剧减少可能是由于蛋白质构象的变化。
We recently reported that the binding of divalent metal ions to yeast enolase (2-phospho-D-glycerate hydrolyase, EC 4.2. 1. l 1) is coupled to proton equilibria [11. This conclusion was based on flow microcalorimetric measurements of the heat of metal binding in three buffers with different heats of ionization. The thermal data were shown to be consistent with the release of two protons when the two, specific cation binding sites on the apoenzyme [2] are filled with Mg” ions at pH 7.5 and 0.05 M ionic strength. The calorimetric experiments could not distinguish between the release of one proton as each Mg*+ ion binds and the release of both protons in response to the conformational change triggered by the binding of the first Mg*’ion [3, 4] Neither could calorimetric measurements at a single pH establish the number or the identity of the titratable groups involved. We report direct measurements of proton release by yeast enolase upon binding Mg*’ions undertaken to clarify these points. The proton release-pH profile is an unsymmetrical bell-shaped curve. The maximum number of protons is released at pH 6.5 and exceeds two, implicating morr: than two titratable groups in metal binding. The shape of the curve above pH 6.5 is consistent with the complexation of metal by histidyl residues. Above pH 7 two Mg*+ ions bind with dissociation constants G 5 PM, and proton release coincides with metal binding. The affinity of enolase for Mg*’ions decreases below pH 7. The sharp decrease in the number of protons released below pH 6.5 may result from a change in the conformation of the protein.