Activase region on chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase - Nonconservative substitution in the large subunit alters species specificity of protein interaction

In the active form of ribulose-1,5 bisphosphate carboxylase/oxygenase (Rubi sco, EC 4.1.1.39), a carbamate at lysine 201 binds Mg2+, which then interac ts with the carboxylation transition state. Rubisco activase facilitates th is spontaneous carbamylation/metal-binding process by removing phosphorylat ed inhibitors from the Rubisco active site. Activase from Solanaceae plants (e.g. tobacco) fails to activate Rubisco from non-Solanaceae plants (e.g. spinach and Chlamydomonas reinhardtii), and non-Solanaceae activase fails t o activate Solanaceae Rubisco. Directed mutagenesis and chloroplast transfo rmation previously showed that a proline 89 to arginine substitution on the surface of the large subunit of Chlamydomonas Rubisco switched its specifi city from non-Solanaceae to Solanaceae activase activation. To define the s ize and function of this putative activase binding region, substitutions we re created at positions flanking residue 89. As in the past, these substitu tions changed the identities of Chlamydomonas residues to those of tobacco. Whereas an aspartate 86 to arginine substitution had little effect, aspart ate 94 to lysine Rubisco was only partially activated by spinach activase b ut now fully activated by tobacco activase. In an attempt to eliminate the activase/tubisco interaction, proline 89 was changed to alanine, which is n ot present in either non-Solanaceae or Solanaceae Rubisco. This substitutio n also caused reversal of activase specificity, indicating that amino acid identity alone does not determine the specificity of the interaction.