Influence of pH on the Mn2+ activation of and binding to yeast enolase: a functional study.
Influence of pH on the Mn2+ activation of and binding to yeast enolase: a functional study.
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pH 对酵母烯醇酶 Mn2 活化和结合的影响：一项功能研究。
DOI：
10.1021/bi00122a038
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发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Nowak,T
中科院分区：
文献类型：
--
作者：
Lee,BH;Nowak,T
Department of Chemistry and Biochemistry，University of Notre Dame，Notre Dame，印第安纳州46556接收于1991年7月19日;修订的Mandarin pt接收于1991年11月1日摘要：通过稳态动力学测量pH对Mn 2+激活酵母烯醇化酶的影响。通过EPR光谱测量了pH对Mn 2+与脱辅基烯醇化酶和烯醇化酶-底物复合物结合的影响。在pH值高于6.6时，Mn 2+的活化符合Michaelis-Menten动力学，但在较高浓度的Mn 2+下，观察到抑制作用。在类似于动力学研究的条件下，酶结合两个Mn2+每二聚体的Kd在微摩尔范围内。在底物2-磷酸甘油酸的存在下，每个单体检测到三个不同的阳离子结合位点，并通过拟合数据确定结合常数。随着pH降低，反应速度降低，阳离子抑制变得最小。在这些条件下，每个单体只有两个Mn~（2+）结合位点，第三个位点一定是抑制位点。速度和动力学常数受缓冲液的影响最小，除了在pH 5.8的PIPES。在这些条件下，速度仅为用其他缓冲液观察到的速度的约40%，并且在存在或不存在底物的情况下，每个单体仅检测到单个Mn 2+结合位点。第二种阳离子在催化机制中的直接作用受到质疑。Mn 2+在站点I的结合常数在7.5至5.2的范围内与pH无关，并且在站点II的结合在相同的pH范围内仅略微增加。这些结果表明，在位置I和II处的阳离子位点含有在该范围内不依赖于pH的配体。位点III可能含有组氨酸，也可能位于底物的磷酰基上。这些基团中的每一个具有6.5至7.0之间的pXa值，并且在低于7.0时成为不良配体。所提出的结果支持的证据，烯醇化酶atcation网站I和II的配体含有氧原子，而不是氮，这两个网站是重要的催化活性。
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556 Received July 19, 1991; Revised Manuscript Received November 1, 1991 abstract: The influence of pH on the activation of yeast enolase by Mn2+ was measured by steady-state kinetics. The pH influence on the binding of Mn2+ to apoenolase and the enolase-substrate complex was measured by EPR spectroscopy. At pH values above 6.6, activation by Mn2+ is fit by Michaelis-Menten kinetics, but at higher concentrations of Mn2+, inhibition is observed. Under conditions analogous to the kinetic studies, the enzyme binds two Mn2+ per dimer with a Kd in the micromolar range. In the presence of the substrate 2-phosphoglycerate, three thermodynamically distinct cation binding sites per monomer are detected and the binding constants are determined by a fit to the data. As the pH decreases, the reaction velocity decreases and the cation inhibition becomes minimal. Under these conditions, only two Mn2+ binding sites per monomerare observed; the third site must be the inhibitory site. The velocity and kinetic constants are minimally affected by buffer except at pH 5.8 with PIPES. Under these conditions, the velocity is only about 40% that observed with other buffers and only a single binding site for Mn2+ per monomer is detected in the presence or absence of substrate. A direct role in the catalytic mechanism by the second cation is called to question. The binding constant for Mn2+ at site I is independent of pH over the range from 7.5 to 5.2, and the binding at site II increases only slightly over this same pH range. These results indicate that the cation sites at positions I and II contain ligands that are pH independent over this range. Site III may contain histidine (s) and may alsobe located at the phosphoryl group of the substrate. Each of these groups has a pXa value between 6.5 and 7.0 and becomes a poor ligand below 7.0. The results presented support the evidence that the ligands on enolase atcation sites I and II contain oxygen atoms, not nitrogen, and that these two sites are important for catalytic activity.
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