Consler T G, Lee J C
E. A. Doisy Department of Biochemistry, St. Louis University School of Medicine, Missouri 63104.
J Biol Chem. 1988 Feb 25;263(6):2787-93.
The structural stability of rabbit muscle pyruvate kinase was examined. The unfolding of pyruvate kinase was induced by guanidine hydrochloride, and the process was monitored by spectroscopic techniques (fluorescence and UV absorption) and hydrodynamic measurements (sedimentation velocity, sedimentation equilibrium, densimetry, and viscometry). The spectroscopic techniques revealed that the unfolding of pyruvate kinase induced by guanidine hydrochloride is not a simple cooperative process. This suggests that different regions of pyruvate kinase are unfolding with different efficiencies in response to the denaturant. These regions are most likely related to the domain structures observed by x-ray crystallography. In the presence of L-phenylalanine, the allosteric inhibitor, the denaturation process became more cooperative, and the enzyme dissociated and unfolded at a higher denaturant concentration. The binding of phenylalanine also induced a structural change in the enzyme, rendering it more susceptible to tryptic digestion. One of the peptides, the production rate of which was increased, was isolated and sequenced. Its N terminus is located at the interface between two domains, one of which contains the active site. This evidence indicates structural changes, probably involving domain-domain interaction, for pyruvate kinase in response to phenylalanine binding.
研究了兔肌肉丙酮酸激酶的结构稳定性。用盐酸胍诱导丙酮酸激酶的去折叠，并通过光谱技术（荧光和紫外吸收）和流体动力学测量（沉降速度、沉降平衡、密度测定和粘度测定）监测该过程。光谱技术表明，盐酸胍诱导的丙酮酸激酶去折叠不是一个简单的协同过程。这表明丙酮酸激酶的不同区域对变性剂的响应以不同效率去折叠。这些区域很可能与X射线晶体学观察到的结构域结构有关。在变构抑制剂L-苯丙氨酸存在下，变性过程变得更具协同性，并且酶在更高的变性剂浓度下解离和去折叠。苯丙氨酸的结合也诱导了酶的结构变化，使其更易被胰蛋白酶消化。分离并测序了其中一个生成速率增加的肽段。其N端位于两个结构域之间的界面处，其中一个结构域包含活性位点。该证据表明，丙酮酸激酶响应苯丙氨酸结合发生了结构变化，可能涉及结构域-结构域相互作用。
