title: Role of isoleucine-164 at the active site of rubisco from Rhodospirillum rubrum.
abstract: Isoleucine-164 is in Van der Waals contact with two ligands (lysine-191 and aspartate-193) of the activator magnesium ion at the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum. To observe the effect of mutations in the second sphere of coordination of the metal ion, isoleucine-164 was replaced by threonine, asparagine, and aspartate. All the mutant enzymes obtained exhibit a low carboxylase activity. Ile164Asp has less than 0.1% of the wild-type carboxylase activity, Ile164Thr and Ile164Asn 6 and 1%, respectively. The mutations increase the Km(RuBP) and decrease the Kcat of the mutated enzymes. The Kcat/Km(RuBP) of Ile164Thr and Ile164Asn are 40- and 900-fold lower than wild-type, respectively. The alteration of the hydrophobic contacts between isoleucine-164 and the metal ion ligands modifies the binding of the magnesium ion and the stabilization of the 2-carboxy-arabinitol 1,5-bisphosphate and decreases the specificity factor, tau.
authors.affiliation: Genencor International, South San Francisco, California 94127, USA.
authors.forename: P
authors.initials: P
authors.lastname: Chène
authors.forename: A G
authors.initials: AG
authors.lastname: Day
authors.forename: A R
authors.initials: AR
authors.lastname: Fersht
mesh_terms: D001665:Binding Sites; D004789:Enzyme Activation; D007532:Isoleucine / Q000235:genetics / Q000502:physiology*; D008274:Magnesium / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000378:metabolism; D012247:Rhodospirillum rubrum / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D007532:Isoleucine; D012273:Ribulose-Bisphosphate Carboxylase; D008274:Magnesium