de Cima Sergio, Rúa Javier, del Valle Pilar, Busto Félix, Baroja-Mazo Alberto, de Arriaga Dolores
Departamento de Biología Molecular, Universidad de León, Campus de Vegazana, 24007 León, Spain.
J Biochem. 2007 Aug;142(2):247-55. doi: 10.1093/jb/mvm126.
The stability of acetyl-CoA synthetases (ACS1 and ACS2) from P. blakesleeanus against temperature, urea and trypsin was studied and compared. Thermal inactivation of ACS1 was biphasic, while that of ACS2 was monophasic. The thermodynamic parameters calculated from the inactivation profiles show ACS2 to be a more thermostable enzyme than ACS1. The presence of ATP and Mg(2+) exerted a protective effect on both enzymes, and led to a marked increase in the E(a), DeltaH(not =), DeltaS(not =) and DeltaG(not =) values. ACS2 is also much more stable against denaturation with urea; the estimates of DeltaG(w) (free energy change for protein unfolding at zero denaturant concentration) were 9.4 kJ mol(-1) and 18.1 kJ mol(-1) for ACS1 and ACS2, respectively. Finally, a half-life of 44.5 min for ACS2 versus the 21 min for ACS1 indicates that ACS2 is more stable than ACS1 against digestion by trypsin. These results seem to show that ACS2 is more rigid overall than ACS1, which may be essential for preserving its catalytic activity in the stress situation in which it is expressed.
研究并比较了来自布氏根霉的乙酰辅酶A合成酶（ACS1和ACS2）对温度、尿素和胰蛋白酶的稳定性。ACS1的热失活是双相的，而ACS2的热失活是单相的。根据失活曲线计算的热力学参数表明，ACS2是比ACS1更耐热的酶。ATP和Mg(2+)的存在对两种酶都有保护作用，并导致E(a)、ΔH(≠)、ΔS(≠)和ΔG(≠)值显著增加。ACS2对尿素变性也更稳定；对于ACS1和ACS2，ΔG(w)（零变性剂浓度下蛋白质解折叠的自由能变化）的估计值分别为9.4 kJ mol(-1)和18.1 kJ mol(-1)。最后，ACS2的半衰期为44.5分钟，而ACS1的半衰期为21分钟，这表明ACS2比ACS1对胰蛋白酶消化更稳定。这些结果似乎表明，ACS2总体上比ACS1更刚性，这对于在其表达的应激情况下保持其催化活性可能至关重要。
