Szilágyi A N, Vas M
Biological Research Center, Institute of Enzymology, Hungarian Academy of Sciences, Budapest.
Biochemistry. 1998 Jun 9;37(23):8551-63. doi: 10.1021/bi973072k.
3-Phosphoglycerate kinase is a typical two-domain "hinge-bending" enzyme, which is known to be regulated by multivalent anions. Here a relationship between this regulation and the hinge-bending domain closure is proposed on the basis of enzyme kinetic analysis and molecular modeling. Activation of the pig muscle enzyme at low concentrations and inhibition at high concentrations of various anionic analogues of the substrate 3-phosphoglycerate or of the nonsubstrate metal-free ATP are described by a two-site model assuming separate sites for activation and inhibition, respectively. Kinetic experiments with various pairs of analogues suggest the presence of a common site for activation by all effectors, separate from the catalytic site for 3-phosphoglycerate; and a common site for inhibition, except for metal-free ATP, identical with the catalytic site of 3-phosphoglycerate. An additional inhibiting site for all of the anions investigated, including metal-free ATP, is also proposed. A similar two-site model can describe activation of the enzyme by a large excess of each substrate; here the ligand binds to the catalytic site as a substrate and to the regulatory site as an activator. Activation is exerted not only by the physiological substrate, 3-phophoglycerate, but also by a synthetic weak substrate. The activity in the reaction with 3-phosphoglycerate and MgATP is greatly enhanced by the simultaneous presence of the weak substrate. This finding clearly proves the existence of a regulatory site, separate from the catalytic site. This regulatory site, however, may only exist in the catalytically competent closed conformation of the enzyme, as indicated by molecular modeling. Docking of the regulator anions into the known X-ray structures of the enzyme revealed the appearance of an anion binding site between the two domains, including the invariant residues of Lys-215 (C-domain) and of Arg-65 among other residues of the basic cluster (N-domain), as a consequence of the large-scale substrate-induced conformational change that leads to domain closure.
3-磷酸甘油酸激酶是一种典型的双结构域“铰链弯曲”酶，已知其受多价阴离子调节。在此，基于酶动力学分析和分子建模，提出了这种调节与铰链弯曲结构域闭合之间的关系。通过一个双位点模型描述了猪肌肉酶在低浓度下被激活以及在高浓度下被底物3-磷酸甘油酸的各种阴离子类似物或无金属ATP抑制的情况，该模型分别假设了激活位点和抑制位点。对各种类似物对进行的动力学实验表明，所有效应物存在一个共同的激活位点，与3-磷酸甘油酸的催化位点分开；除无金属ATP外，存在一个共同的抑制位点，与3-磷酸甘油酸的催化位点相同。还提出了一个针对所有研究阴离子（包括无金属ATP）的额外抑制位点。一个类似的双位点模型可以描述每种底物大量过量时酶的激活情况；在此，配体作为底物结合到催化位点，作为激活剂结合到调节位点。激活不仅由生理底物3-磷酸甘油酸发挥作用，还由一种合成的弱底物发挥作用。同时存在弱底物时，与3-磷酸甘油酸和MgATP反应的活性会大大增强。这一发现清楚地证明了存在一个与催化位点分开的调节位点。然而，如分子建模所示，这个调节位点可能仅存在于酶具有催化活性的闭合构象中。将调节阴离子对接至酶的已知X射线结构中，揭示了在两个结构域之间出现一个阴离子结合位点，包括Lys-215（C结构域）的不变残基以及碱性簇（N结构域）其他残基中的Arg-65，这是由导致结构域闭合的大规模底物诱导构象变化所导致的。
