Violet M, Meunier J C
Laboratoire de Chimie Biologique, Institut National Agronomique, Thiverval-Grignon, France.
Biochem J. 1989 Nov 1;263(3):665-70. doi: 10.1042/bj2630665.
The irreversible thermal inactivation of Bacillus licheniformis alpha-amylase was studied. A two-step behaviour in the irreversible denaturation process was found. Our experimental results are consistent only with the two-step model and rule out the two-isoenzyme one. They suggest that the deactivation mechanism involves the existence of a temperature-dependent intermediate form. Therefore the enzyme could exist in a great number of active conformational states. We have shown that Ca2+ is necessary for the structural integrity of alpha-amylase. Indeed, dialysis against chelating agents leads to a reversible enzyme inactivation, though molecular sieving has no effect. Further, the key role of Ca2+ in the alpha-amylase thermostability is reported. The stabilizing effect of Ca2+ is reflected by the decrease of the denaturation constants of both the native and the intermediate forms. Below 75 degrees C, in the presence of 5 mM-CaCl2, alpha-amylase is completely thermostable. Neither other metal ions nor substrate have a positive effect on enzyme thermostability. The effect of temperature on the native enzyme and on one intermediate form was studied. Both forms exhibit the same optimum temperature. Identical activation parameters for the hydrolytic reaction catalysed by these two forms were found.
研究了地衣芽孢杆菌α-淀粉酶的不可逆热失活。在不可逆变性过程中发现了两步行为。我们的实验结果仅与两步模型一致，并排除了双同工酶模型。结果表明，失活机制涉及一种温度依赖性中间形式的存在。因此，该酶可能以大量活性构象状态存在。我们已经表明，Ca2+对于α-淀粉酶的结构完整性是必需的。实际上，用螯合剂透析会导致酶的可逆失活，而分子筛则没有影响。此外，还报道了Ca2+在α-淀粉酶热稳定性中的关键作用。Ca2+的稳定作用通过天然形式和中间形式的变性常数的降低来体现。在75℃以下，在5 mM CaCl2存在下，α-淀粉酶完全热稳定。其他金属离子和底物对酶的热稳定性均无积极影响。研究了温度对天然酶和一种中间形式的影响。两种形式表现出相同的最适温度。发现这两种形式催化的水解反应具有相同的活化参数。
