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The characterization of mutant Bacillus subtilis adenylosuccinate lyases corresponding to severe human adenylosuccinate lyase deficiencies
Author(s) -
Palenchar Jennifer Brosius,
Crocco Jennifer M.,
Colman Roberta F.
Publication year - 2003
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.0303903
Subject(s) - biology , biochemistry , lyase , complementation , bacillus subtilis , homotetramer , microbiology and biotechnology , enzyme , mutant , protein subunit , gene , genetics , bacteria
Adenylosuccinate lyase is a homotetramer that catalyzes two discrete reactions in the de novo synthesis of purines: the cleavage of adenylosuccinate and succinylaminoimidazole carboxamide ribotide (SAICAR). Several point mutations in the gene encoding the enzyme have been implicated in human disease. Bacillus subtilis adenylosuccinate lyase was used as a model system in which mutations were constructed corresponding to those mutations associated with severe human adenylosuccinate lyase deficiency. Site‐directed mutagenesis was utilized to construct amino acid substitutions in B. subtilis adenylosuccinate lyase; Met 10 , Ile 123 , and Thr 367 were replaced by Leu, Trp, and Arg, respectively, and the altered enzymes were expressed in Escherichia coli . These purified enzymes containing amino acid substitutions were found to have substantial catalytic activity and exhibit relatively small changes in their kinetic parameters. The major deviations from the wild‐type‐like behavior were observed upon biophysical characterization. All of these enzymes with amino acid replacements are associated with marked thermal instability. I123W adenylosuccinate lyase exhibits notable changes in the circular dichroism spectra, and a native gel electrophoresis pattern indicative of some protein aggregation. T367R also exhibits alterations at the quarternary level, as reflected in native gel electrophoresis. Experimental results, combined with homology modeling, suggest that the altered enzymes are primarily structurally impaired. The enzyme instability was found to be lessened by subunit complementation with the wild‐type enzyme, under mild conditions; these studies may have implications for the in vivo behavior of adenylosuccinate lyase in heterozygous patients. Residues Met 10 , Ile 123 , and Thr 367 appear to be located in regions of the enzyme important for maintaining the structural integrity required for a stable, functional enzyme.