Evaluation of types of interactions in subunit association in Bacillus subtilis adenylosuccinate lyase.
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.
De Zoysa Ariyananda
Lushanti
L
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, USA.
Colman
Roberta F
RF
D000264:Adenylosuccinate Lyase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D002942:Circular Dichroism; D003198:Computer Simulation; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D013696:Temperature
D021122:Protein Subunits; D000264:Adenylosuccinate Lyase