title: In vitro hybridization and separation of hybrids of human adenylosuccinate lyase from wild-type and disease-associated mutant enzymes.
abstract: Human adenylosuccinate lyase (ASL) deficiency is an inherited metabolic disease in which the majority of the patients are compound heterozygotes for the mutations that occur in the ASL gene. Starting with purified wild-type (WT) and single-mutant human ASL, we generated in vitro hybrids that mimic compound heterozygote ASL. For this study, we used His-tagged WT/non-His-tagged WT, His-tagged WT/non-His-tagged R396C, His-tagged WT/non-His-tagged R396H, His-tagged R194C/non-His-tagged R396C, and His-tagged L311V/non-His-tagged R396H enzyme pairs. We generated various hybrids by denaturing pairs of enzymes in 1 M guanidinium chloride and renaturing them by removing the denaturant. The hybrids were separated on a nickel-nitrilotriacetic acid-agarose column based on the number of His tags present in the enzyme tetramer. Analytical ultracentrifuge data indicate that the hybrids have predominant amounts of heterotetramers. Analysis of the V(max) values of the hybrids indicates that most of the subunits behave independently; however, the hybrid tetramers retain weak positive cooperativity, indicating that there is some interaction between the different subunit types. The interactions between WT and mutant subunits may be advantageous to the parents of ASL deficient patients, while the interactions between some mutant subunits may assist heterozygote ASL deficient patients.
authors.affiliation: Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA.
authors.forename: Lushanti
authors.initials: L
authors.lastname: De Zoysa Ariyananda
authors.forename: Christina
authors.initials: C
authors.lastname: Antonopoulos
authors.forename: Jenna
authors.initials: J
authors.lastname: Currier
authors.forename: Roberta F
authors.initials: RF
authors.lastname: Colman
mesh_terms: D000264:Adenylosuccinate Lyase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives / Q000378:metabolism; D019540:Area Under Curve; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006639:Histidine; D006801:Humans; D007700:Kinetics; D008661:Metabolism, Inborn Errors / Q000201:enzymology / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D009154:Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D015202:Protein Engineering / Q000379:methods*; D020673:Protein Renaturation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012265:Ribonucleotides / Q000378:metabolism; D019802:Succinic Acid / Q000737:chemistry / Q000378:metabolism; D013696:Temperature
chemical_list: D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; D012265:Ribonucleotides; D000620:Aminoimidazole Carboxamide; D006639:Histidine; D019802:Succinic Acid; D000264:Adenylosuccinate Lyase; C020635:SAICAR