title: Characterization of a mutant Bacillus subtilis adenylosuccinate lyase equivalent to a mutant enzyme found in human adenylosuccinate lyase deficiency: asparagine 276 plays an important structural role.
abstract: Adenylosuccinate lyase, an enzyme catalyzing two reactions in purine biosynthesis (the cleavage of either adenylosuccinate or succinylaminoimidazole carboxamide ribotide), has been implicated in a human disease arising from point mutations in the gene encoding the enzyme. Asn(276) of Bacillus subtilis adenylosuccinate lyase, a residue corresponding to the location of a human enzyme mutation, was replaced by Cys, Ser, Ala, Arg, and Glu. The mutant enzymes exhibit decreased V(max) values (2-400-fold lower) for both substrates compared to the wild-type enzyme and some changes in the pH dependence of V(max) but no loss in affinity for adenylosuccinate. Circular dichroism reveals no difference in secondary structure between the wild-type and mutant enzymes. We show here for the first time that wild-type adenylosuccinate lyase exhibits a protein concentration dependence of molecular weight, secondary structure, and specific activity. An equilibrium constant between the dimer and tetramer was measured by light scattering for the wild-type and mutant enzymes. The equilibrium is somewhat shifted toward the tetramer in the mutant enzymes. The major difference between the wild-type and mutant enzymes appears to be in quaternary structure, with many mutant enzymes exhibiting marked thermal instability relative to the wild-type enzyme. We propose that mutations at position 276 result in structurally impaired adenylosuccinate lyases which are assembled into defective tetramers.
authors.affiliation: Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, USA.
authors.forename: Jennifer Brosius
authors.initials: JB
authors.lastname: Palenchar
authors.forename: Roberta F
authors.initials: RF
authors.lastname: Colman
mesh_terms: D000249:Adenosine Monophosphate / Q000031:analogs & derivatives* / Q000737:chemistry; D000264:Adenylosuccinate Lyase / Q000737:chemistry* / Q000172:deficiency* / Q000235:genetics / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives* / Q000737:chemistry; D001120:Arginine / Q000235:genetics; D001216:Asparagine / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D002942:Circular Dichroism; D004789:Enzyme Activation / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017433:Protein Structure, Secondary / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012265:Ribonucleotides / Q000737:chemistry; D013379:Substrate Specificity / Q000235:genetics; D013912:Threonine / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D012265:Ribonucleotides; C012168:adenylosuccinate; D013912:Threonine; D000620:Aminoimidazole Carboxamide; D000249:Adenosine Monophosphate; D001216:Asparagine; D001120:Arginine; D000264:Adenylosuccinate Lyase; C020635:SAICAR