title: Analysis of allosteric effector binding sites of potato ADP-glucose pyrophosphorylase through reverse genetics.
abstract: ADP-glucose pyrophosphorylase (AGPase) is a key regulatory enzyme of bacterial glycogen and plant starch synthesis as it controls carbon flux via its allosteric regulatory behavior. Unlike the bacterial enzyme that is composed of a single subunit type, the plant AGPase is a heterotetrameric enzyme (alpha2beta2) with distinct roles for each subunit type. The large subunit (LS) is involved mainly in allosteric regulation through its interaction with the catalytic small subunit (SS). The LS modulates the catalytic activity of the SS by increasing the allosteric regulatory response of the hetero-oligomeric enzyme. To identify regions of the LS involved in binding of effector molecules, a reverse genetics approach was employed. A potato (Solanum tuberosum L.) AGPase LS down-regulatory mutant (E38A) was subjected to random mutagenesis using error-prone polymerase chain reaction and screened for the capacity to form an enzyme capable of restoring glycogen production in glgC(-) Escherichia coli. Dominant mutations were identified by their capacity to restore glycogen production when the LS containing only the second site mutations was co-expressed with the wild-type SS. Sequence analysis showed that most of the mutations were decidedly nonrandom and were clustered at conserved N- and C-terminal regions. Kinetic analysis of the dominant mutant enzymes indicated that the K(m) values for cofactor and substrates were comparable with the wild-type AGPase, whereas the affinities for activator and inhibitor were altered appreciably. These AGPase variants displayed increased resistance to P(i) inhibition and/or greater sensitivity toward 3-phosphoglyceric acid activation. Further studies of Lys-197, Pro-261, and Lys-420, residues conserved in AGPase sequences, by site-directed mutagenesis suggested that the effectors 3-phosphoglyceric acid and P(i) interact at two closely located binding sites.
authors.affiliation: Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164, USA.
authors.forename: I H
authors.initials: IH
authors.lastname: Kavakli
authors.forename: J S
authors.initials: JS
authors.lastname: Park
authors.forename: C J
authors.initials: CJ
authors.lastname: Slattery
authors.forename: P R
authors.initials: PR
authors.lastname: Salamone
authors.forename: J
authors.initials: J
authors.lastname: Frohlick
authors.forename: T W
authors.initials: TW
authors.lastname: Okita
mesh_terms: D000495:Allosteric Site; D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D051599:Glucose-1-Phosphate Adenylyltransferase; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010641:Phenotype; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D011198:Solanum tuberosum / Q000201:enzymology*
chemical_list: D017931:DNA Primers; D009713:Nucleotidyltransferases; D051599:Glucose-1-Phosphate Adenylyltransferase