The effect of genetic single point mutation Q92P of HCA Ⅱ which is located near the activity site upon the activity and stability was investigated.Although not directly involved in the HCA Ⅱ catalysis,this mutation impacted on HCA Ⅱ activity,stability and folding process.The activity of mutant was dramatically reduced since the simulation results exhibited that the hydrogen bonds at the specific site were altered.Spectroscopic surveys revealed that the mutation leaded more buried Trp residues to be exposed to external surroundings but did not seriously affect the secondary structure or the hydrophobic exposure.The mutant was less stable than the pseudo-wild type protein against chemical-induced denaturation.The mutation slightly decreased the stability of the molten globular intermediate,but to a greater extent affected the stability of the native state by an approximately 80％ reduction of the Gibbs free energy for the transition from the native state to the intermediate.This also may be conducive to explain the existence of accumulated aggregation-prone molten globular intermediate which further affectclassical pathway of refolding.The results suggested that the Q92 may be important for the enzyme activity and stability.%本文研究了单点突变Q92P对酶活性,稳定性以及折叠途径的影响.尽管该位点不直接参与底物催化,但92位氨基酸的突变对酶的活性,稳定性以及折叠过程产生了重大的影响.该突变的酶活性有较大程度的降低,通过模拟发现该位点的氢键网被破坏.而且光谱实验表明该突变导致更多被掩埋的色氨酸暴露于周边环境,但并没有较大的影响到二级结构和疏水面暴露程度.在对抗化学品诱导的去折叠程度上,相比于野生型蛋白,该突变蛋白稳定性较差.通过吉布斯自由能的计算发现该突变位点轻微的降低了中间体的稳定性,但相对更大的影响了天然态的稳定性,天然态至中间体的能量大约为野生型的20％左右.该结果有组于解释在折叠过程中倾向于聚沉的中间体的存在,该中间体的存在影响了正常的折叠路径.该实验表明92位氨基酸对人碳酸酐酶Ⅱ的活性和稳定性很重要.
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