Ribulosebisphosphate carboxylase: amino acid sequence of a peptide bearing the activator carbon dioxide

Abstract

Ribulosebisphosphate carboxylase is activated by reaction of an activator CO2 to form a carbamate on the epsilon-amino group of a lysyl residue on the large catalytic subunit. This carbamate has been converted to the methoxycarbonyl derivative by treatment of the enzyme with diazomethane as previously reported [Lorimer, G. H., & Miziorko, H. H. (1980) Biochemistry 19, 5321]. Digestion of the methylated enzyme--14CO2 complex with trypsin yielded several radioactive peptides which were purified by using standard chromatographic procedures. Sequence analyses revealed that these peptides had the same sequence: -Gly-Gly-Leu-Asp-Phe5-Thr-Lys-Asp-Asp-Glu10-Asn-Val-Asn-Ser-Gln15-Pro-Phe. Residue 7 was 14C labeled and emerged from the sequencer as the phenylthiohydantoin derivative of N epsilon-(methoxycarbonyl)lysine. The acidic nature of the residues close to the lysine bearing the activator CO2 provides a molecular explanation for the pH and divalent metal ion dependency of the activation reaction. An entirely homologous sequence has been found in the large subunit of the enzyme from Zea mais [McIntosh, L., Poulsen, C., & Bogorad, L. (1980) Nature (London) 288, 556]. The lysyl residue bearing the activator CO2 is 26 residues removed from one of the lysyl residues identified by use of the affinity label N-bromoacetyl-ethanolamine phosphate as being within the active-site domain.