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Evaluation of Types of Interactions in Subunit Association in Bacillus subtilis Adenylosuccinate Lyase (ASL)
Author(s) -
De Zoysa Ariyananda Lushanti,
Colman Roberta
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a641-a
Subject(s) - bacillus subtilis , protein subunit , biology , chemistry , genetics , bacteria , gene
Adenylosuccinate lyase of B. 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, in the presence of KBr and after mutagenesis. Analytical ultracentrifugation data at 8°and 4 °Creveal a decrease in the weight average molecular weight to ~150 kD from the initial value of 200 kDa at 25 °C. In contrast, the enzyme dissociates successively to a trimer, a dimer (100 kDa) and a monomer (50 kDa) in the presence of increasing KBr concentrations (0.1–3M). Very low enzymatic activity was found under these conditions. Accordingly, we postulate that electrostatic interactions are the major source of oligomeric stabilization. We selected for mutagenesis the closest charged residues (H299/E239 and R167/D217 pairs) located in the subunit interface that has the largest surface area. All the mutants have very low V max values, high K m values and decreased molecular weights. The R167/D217 pair makes the greater contribution to the stability of the oligomer. These results support the proposed importance of electrostatic interactions in maintaining the ASL tetramer and indicate that this structure is essential for adenylosuccinate lyase activity. (Supp. by NIH DK60504)