Distance between two active-site lysines of ribulose bisphosphate carboxylase from Rhodospirillum rubrum.

In the absence of a three-dimensional structure of ribulose-bisphosphate carboxylase/oxygenase [3-phospho-D-glycerate carboxy-lyase(dimerizing), EC 4.1.1.39], we have probed the distance between two active-site lysyl residues (Lys-166 and Lys-329) of the Rhodospirillum rubrum enzyme with 4,4'-diisothiocyano-2,2'-disulfonate stilbene, a covalent cross-linking reagent that spans 12 A. The reagent rapidly inactivated the carboxylase, and a competitive inhibitor provided substantial protection. To remove products arising from intersubunit or intermolecular cross-linking, the inactivated enzyme was subjected to gel filtration in the presence of urea. Inspection of a tryptic digest of the isolated monomeric fraction revealed that more than half of the incorporated reagent was associated with a single peptide. This peptide was purified by gel filtration, followed by high HPLC. Compositional and sequence analyses of the purified peptide established that it was composed of two chains, encompassing positions 149-168 and 314-337 of the original protein subunit and connected by a cross-link between Lys-166 and Lys-329. Thus, the two active-site lysines of the carboxylase can be juxtaposed within 12 A, a finding that is consistent with their purported proximity to ribulose bisphosphate in the enzyme-substrate complex. The cross-link was not formed when the carboxylase was treated with the reagent either in the presence of a transition-state analogue (carboxyarabinitol bisphosphate) or in the absence of CO(2) and Mg(2+), conditions under which the enzyme exists in a deactivated form.