Ribulosebisphosphate carboxylase/oxygenase : synthesis of potential affinity labels and characterization of active-site regions

Ribulosebisphosphate carboxylase/oxygenase catalyzes both the carboxylation and oxidative cleavage of ribulose 1,5-bisphosphate, opposing reactions in photosynthetic carbon assimilation and photorespiration, respectively. One approach to understanding this bifunctionality entails identification of active-site regions of the enzyme by affinity labeling. Comparison of active-site residues in enzymes from evolutionarily distant should reveal conserved molecular features which are likely to sources function in catalysis.

Irregularities in a published report from another laboratory prompted the reexamination of the binding site for pyridoxal 5'-phosphate in the carboxylase from Rhodospirillum rubrum, described in Part I. A single pyridoxal 5'phosphate-labeled peptide was obtained. The reactive lysyl residue lies in a seven-residue sequence common to all known carboxylases.

Part II describes the synthesis and characterization of four potential affinity labels for the carboxylase. Reductive amination of ribulose 1,5-bisphosphate with bifunctional amines by cyanoborohydride and bromoacetylation of the isolated amino bisphosphates gave 2-(4-bromoacetamido)anilino-2-deoxypentitol 1,5-bisphosphate (reagents A and B, epimeric at C2), 2-[2-(bromoacetamido)ethyl]amino-2-deoxypentitol 1,5- bisphophate (reagent C), 2-[4-(bromoacetamido)butyl]amino-2-deoxypentitol 1,5-bisphosphate (reagent D), and 2-{4-[4-(bromoacetamido)phenyl methyl]- anilino}-2-deoxypentitol 1,5-bisphosphate (reagent E).

The interactions of the epimeric reagents A and B with the carboxylase consistent with the documented effect of configuration at C2 on Reductive amination of were binding to the enzyme, Of the two, only reagent B Irreversibly inactivated the R. rubrum enzyme in a time-dependent manner, of reagent B-modified tryptic peptides revealed two approximately equal sites of labeling, His⁴⁴ and Cys⁵⁸ both of which were subject to protection by 2-carboxyribitol 1,5-bisphosphate. Although these residues are not conserved in the six known carboxylase sequences, they lie in a Isolation region of homology not previously known to be at or near the active site of the enzyme.

Reagents C and D also fulfilled criteria of affinity labeling; the former alkylated Met³³⁵ while the latter alkylated His⁴⁴, Cys⁵⁵, and Met³³⁵. The methionyl residue in the R. rubrum enzyme, previously modified by two affinity labels, is replaced by leucine in the other carboxylase. Reagent E, although a competitive inhibitor, exhibited no time-dependent effect on carboxylase activity. The implications of these findings to the active-site geometry of the carboxylase are discussed