Pop Cristina, Feeney Brett, Tripathy Ashutosh, Clark A Clay
Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, North Carolina 27695-7622, USA.
Biochemistry. 2003 Oct 28;42(42):12311-20. doi: 10.1021/bi034999p.
The interface of the procaspase-3 dimer plays a critical role in zymogen maturation. We show that replacement of valine 266, the residue at the center of the procaspase-3 dimer interface, with glutamate resulted in an increase in enzyme activity of approximately 60-fold, representing a pseudoactivation of the procaspase. In contrast, substitution of V266 with histidine abolished the activity of the procaspase-3 as well as that of the mature caspase. While the mutations do not affect the dimeric properties of the procaspase, we show that the V266E mutation may affect the formation of a loop bundle that is important for stabilizing the active site. In contrast, the V266H mutation affects the positioning of loop L3, the loop that forms the bulk of the substrate binding pocket. In some cases, the amino acids affected by the mutations are >20 A from the interface. Overall, the results demonstrate that the integrity of the dimer interface is important for maintaining the proper active site conformation.
半胱天冬酶原-3二聚体的界面在酶原成熟过程中起着关键作用。我们发现，将半胱天冬酶原-3二聚体界面中心的缬氨酸266替换为谷氨酸后，酶活性增加了约60倍，这代表了半胱天冬酶原的假激活。相反，用组氨酸替代V266消除了半胱天冬酶原-3以及成熟半胱天冬酶的活性。虽然这些突变不影响半胱天冬酶原的二聚体性质，但我们发现V266E突变可能会影响对稳定活性位点很重要的环束的形成。相反，V266H突变影响环L3的定位，环L3构成了底物结合口袋的大部分。在某些情况下，受突变影响的氨基酸距离界面>20埃。总体而言，结果表明二聚体界面的完整性对于维持正确的活性位点构象很重要。
