Peterson K M, Srivastava D K
Department of Biochemistry and Molecular Biology, North Dakota State University, Fargo, North Dakota 58105, USA.
Biochemistry. 2000 Oct 17;39(41):12678-87. doi: 10.1021/bi001317e.
The substitution of the C=O by the C=S group in 2-azaoctanoyl-CoA increases the volume of the ligand by 11 A(3), and the excision of a methylene group from Glu-376, via Glu-376 --> Asp (E376D) mutation in medium chain acyl-CoA dehydrogenase (MCAD), creates a complementary cavity of 18 A(3) dimension, just opposite to the ligand's carbonyl group. We investigated whether the newly created cavity would facilitate accommodation of the bulkier (C=O --> C=S substituted) ligand within the active site of the enzyme. To ascertain this, we determined the binding affinity and kinetics of association and dissociation of 2-azaoctanoyl-CoA and the C=O --> C=S substituted ligand, 2-azadithiooctanoyl-CoA, involving the wild-type and Glu-376 --> Asp mutant enzymes. The experimental data revealed that the binding of 2-azadithiooctanoyl-CoA to the wild-type enzyme was energetically unfavorable as compared to 2-azaoctanoyl-CoA. However, such an energetic constraint was alleviated for the binding of the former ligand to the E376D mutant enzyme site. A detailed account of the free energy and enthalpic profiles for the binding of 2-azaoctanoyl-CoA and 2-azadithiooctanoyl-CoA to the wild-type and Glu-376 --> Asp mutant enzymes throws light on the flexibility of the enzyme site cavity in stabilizing the ground and transition states of the enzyme-ligand complexes.
在2-氮杂辛酰辅酶A中，用C=S基团取代C=O会使配体体积增加11 ų，并且通过中链酰基辅酶A脱氢酶（MCAD）中的Glu-376→Asp（E376D）突变从Glu-376切除一个亚甲基，会形成一个尺寸为18 ų的互补腔，正好与配体的羰基相对。我们研究了新形成的腔是否会促进体积更大（C=O→C=S取代）的配体在酶活性位点内的容纳。为了确定这一点，我们测定了2-氮杂辛酰辅酶A和C=O→C=S取代的配体2-氮杂二硫辛酰辅酶A与野生型和Glu-376→Asp突变酶的结合亲和力以及结合和解离动力学。实验数据表明，与2-氮杂辛酰辅酶A相比，2-氮杂二硫辛酰辅酶A与野生型酶的结合在能量上是不利的。然而，对于前一种配体与E376D突变酶位点的结合，这种能量限制得到了缓解。对2-氮杂辛酰辅酶A和2-氮杂二硫辛酰辅酶A与野生型和Glu-376→Asp突变酶结合的自由能和焓变曲线的详细分析揭示了酶活性位点腔在稳定酶-配体复合物的基态和过渡态方面的灵活性。
