Street I P, Withers S G
Department of Chemistry, University of British Columbia, Vancouver, Canada.
Biochem J. 1995 Jun 15;308 ( Pt 3)(Pt 3):1017-23. doi: 10.1042/bj3081017.
The ionization state of the substrate alpha-D-glucopyranosyl phosphate bound at the active site of glycogen phosphorylase has been probed by a number of techniques. Values of Ki determined for a series of substrate analogue inhibitors in which the phosphate moiety bears differing charges suggest that the enzyme will bind both the monoanionic and dianionic substrates with approximately equal affinity. These results are strongly supported by 31P- and 19F-NMR studies of the bound substrate analogues alpha-D-glucopyranosyl 1-methylenephosphonate and 2-deoxy-2-fluoro-alpha-D-glucopyranosyl phosphate, which also suggest that the substrate can be bound in either ionization state. The pH-dependences of the inhibition constants K1 for these two analogues, which have substantially different phosphate pK2 values (7.3 and 5.9 respectively), are found to be essentially identical with the pH-dependence of K(m) values for the substrate, inhibition decreasing according to an apparent pKa value of 7.2. This again indicates that there is no specificity for monoanion or dianion binding and also reveals that binding is associated with the uptake of a proton. As the bound substrate is not protonated, this proton must be taken up by the proton.
已通过多种技术探究了结合在糖原磷酸化酶活性位点的底物α-D-吡喃葡萄糖基磷酸的电离状态。对于一系列磷酸部分带有不同电荷的底物类似物抑制剂所测定的Ki值表明，该酶将以大致相等的亲和力结合单阴离子和双阴离子底物。结合的底物类似物α-D-吡喃葡萄糖基1-亚甲基膦酸酯和2-脱氧-2-氟-α-D-吡喃葡萄糖基磷酸的31P和19F-NMR研究有力地支持了这些结果，这些研究还表明底物可以以任何一种电离状态结合。发现这两种类似物的抑制常数K1的pH依赖性与底物的K(m)值的pH依赖性基本相同，这两种类似物的磷酸pK2值（分别为7.3和5.9）有很大差异，抑制作用根据表观pKa值7.2降低。这再次表明对单阴离子或双阴离子结合没有特异性，并且还揭示结合与质子的摄取有关。由于结合的底物没有质子化，这个质子必须被质子接受体摄取。
