Thomas T M, Scopes R K
School of Biochemistry, La Trobe University, Bundoora, Melbourne, Victoria 3083, Australia.
Biochem J. 1998 Mar 15;330 ( Pt 3)(Pt 3):1087-95. doi: 10.1042/bj3301087.
The effects of temperature on the kinetic parameters kcat and Km, for three isolates of the highly conserved monomeric enzyme 3-phosphoglycerate kinase (PGK), were investigated in detail using a rapid automated kinetics apparatus. PGK was purified from the thermophilic bacterium Thermoanaerobacter sp. Rt8.G4 (optimum growth temperature 68 degrees C), the mesophile Zymomonas mobilis (optimum growth temperature 32 degrees C) and a second, unidentified, soil mesophile designated unid A (optimum growth temperature 27 degrees C). The kinetic behaviour with temperature of each PGK preparation was distinct, despite the conserved nature of the enzyme. The kcat values increased with temperature, but not as rapidly exponentially, as might be expected from the Arrhenius equation. Maximum kcat values were at much higher temperatures than the optimum growth temperatures for the mesophiles, but for the thermophile the temperature of maximum kcat was close to its optimum growth temperature. Km values were in general nearly constant through the lower temperature ranges, but increased substantially as the optimum temperature (highest kcat) was passed. Thermal irreversible denaturation of the PGK proteins was also investigated by measuring loss of activity over time. In a dilute buffer, Arrhenius plots for denaturation were linear, and the calculated apparent energy of activation (Eact) for denaturation for the thermophilic PGK was 600 kJ.mol-1, whereas for the mesophilic enzymes the values were 200-250 kJ.mol-1. In the presence of substrates, a considerable stabilization occurred, and in the case of the Z. mobilis enzyme, the apparent Eact was increased to 480 kJ.mol-1. A theoretical explanation for these observations is presented. Comparing the kinetics data with irreversible denaturation rates determined at relevant temperatures, it was clear that kcat values reached a maximum, and then decreased with higher temperature before irreversible denaturation had any significant influence.
使用快速自动动力学仪器，详细研究了温度对高度保守的单体酶3 - 磷酸甘油酸激酶（PGK）的三种分离株的动力学参数kcat和Km的影响。PGK分别从嗜热细菌嗜热栖热菌Rt8.G4（最适生长温度68℃）、嗜温菌运动发酵单胞菌（最适生长温度32℃）以及另一种未鉴定的土壤嗜温菌unid A（最适生长温度27℃）中纯化得到。尽管该酶具有保守性，但每种PGK制剂的动力学行为随温度变化各不相同。kcat值随温度升高而增加，但不像从阿伦尼乌斯方程预期的那样呈指数快速增加。最大kcat值出现的温度远高于嗜温菌的最适生长温度，但对于嗜热菌，最大kcat值对应的温度接近其最适生长温度。Km值在较低温度范围内通常几乎保持恒定，但在超过最适温度（最高kcat）时会大幅增加。还通过测量活性随时间的损失来研究PGK蛋白的热不可逆变性。在稀缓冲液中，变性的阿伦尼乌斯图呈线性，嗜热PGK变性的计算表观活化能（Eact）为600 kJ·mol-1，而嗜温酶的值为200 - 250 kJ·mol-1。在有底物存在时，会发生显著的稳定作用，对于运动发酵单胞菌的酶，表观Eact增加到480 kJ·mol-1。文中给出了这些观察结果的理论解释。将动力学数据与在相关温度下测定的不可逆变性速率进行比较，很明显kcat值先达到最大值，然后在不可逆变性产生任何显著影响之前随着温度升高而下降。
