De Zoysa Ariyananda Lushanti, Colman Roberta F
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, USA.
Biochemistry. 2008 Mar 4;47(9):2923-34. doi: 10.1021/bi701400c. Epub 2008 Feb 1.
Adenylosuccinate lyase (ASL) of Bacillus subtilis is a homotetramer in which three subunits contribute to each of four active sites. We sought to evaluate the types of interactions responsible for subunit association by studying the enzyme's oligomeric structure at low temperatures as compared to 25 degrees C, in the presence of KBr and after mutagenesis. Analytical ultracentrifugation data reveal that at 25 degrees C ASL is active and exists as 100% tetramer, while at 8 and 4 degrees C, as hydrophobic interactions are weakened, the catalytic activity decreases strikingly and the enzyme dissociates to a mixture of monomer-dimer-trimer, with small amounts of tetramer. In the presence of increasing concentrations of KBr (0.1-2.5 M), which disrupts electrostatic interactions, ASL is dissociated initially to monomer-dimer, with small amounts of trimer-tetramer, and then the monomer species predominates along with small amounts of trimer-tetramer. Very low enzymatic activity was found under these conditions. Accordingly, we postulate that electrostatic interactions are a major source of oligomeric stabilization of B. subtilis ASL. We selected for mutagenesis the closest charged residues (His (299)/Glu (239) and Arg (167)/Asp (217) pairs) located in the subunit interface that has the largest surface area. All of the mutants have low V max values, high K M values, and decreased molecular masses. We conclude that both hydrophobic and electrostatic interactions play roles in maintaining the ASL tetramer and this structure is essential for adenylosuccinate lyase activity.
枯草芽孢杆菌的腺苷酸琥珀酸裂解酶（ASL）是一种同四聚体，其中三个亚基对四个活性位点中的每一个都有贡献。我们试图通过研究该酶在低温（与25℃相比）、存在KBr时以及诱变后的寡聚结构，来评估负责亚基缔合的相互作用类型。分析超速离心数据显示，在25℃时ASL具有活性，以100%的四聚体形式存在，而在8℃和4℃时，由于疏水相互作用减弱，催化活性显著降低，酶解离为单体-二聚体-三聚体的混合物，还有少量四聚体。在存在浓度不断增加的KBr（0.1 - 2.5 M）时，KBr会破坏静电相互作用，ASL最初解离为单体-二聚体，有少量三聚体-四聚体，然后单体形式占主导，还有少量三聚体-四聚体。在这些条件下发现酶活性非常低。因此，我们推测静电相互作用是枯草芽孢杆菌ASL寡聚体稳定的主要来源。我们选择位于具有最大表面积的亚基界面中最接近的带电荷残基（His(299)/Glu(239)和Arg(167)/Asp(217)对）进行诱变。所有突变体的Vmax值都很低，Km值很高，分子量降低。我们得出结论，疏水相互作用和静电相互作用都在维持ASL四聚体中起作用，并且这种结构对于腺苷酸琥珀酸裂解酶活性至关重要。
