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 (Rubisco, EC ), a carbamate at lysine 201 binds Mg(2+), which then interacts with the carboxylation transition state. Rubisco activase facilitates this spontaneous carbamylation/metal-binding process by removing phosphorylated 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 to activate Solanaceae Rubisco. Directed mutagenesis and chloroplast transformation previously showed that a proline 89 to arginine substitution on the surface of the large subunit of Chlamydomonas Rubisco switched its specificity from non-Solanaceae to Solanaceae activase activation. To define the size and function of this putative activase binding region, substitutions were created at positions flanking residue 89. As in the past, these substitutions changed the identities of Chlamydomonas residues to those of tobacco. Whereas an aspartate 86 to arginine substitution had little effect, aspartate 94 to lysine Rubisco was only partially activated by spinach activase but now fully activated by tobacco activase. In an attempt to eliminate the activase/Rubisco interaction, proline 89 was changed to alanine, which is not present in either non-Solanaceae or Solanaceae Rubisco. This substitution also caused reversal of activase specificity, indicating that amino acid identity alone does not determine the specificity of the interaction.
Ott
C M
CM
Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588, USA.
Smith
B D
BD
Portis
A R
AR
Spreitzer
R J
RJ
D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000201:enzymology; D002736:Chloroplasts / Q000201:enzymology; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010947:Plants, Toxic; D011485:Protein Binding; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013045:Species Specificity; D018724:Spinacia oleracea / Q000201:enzymology; D013329:Structure-Activity Relationship; D010959:Tissue Plasminogen Activator / Q000378:metabolism*; D014026:Nicotiana / Q000201:enzymology
D010959:Tissue Plasminogen Activator; D012273:Ribulose-Bisphosphate Carboxylase