Role of Isoleucine-164 at the Active Site of Rubisco fromRhodospirillum rubrum

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.