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Metabolism and Enzymology

Photoaffinity Labeling of Mature and Precursor Forms of the Small Subunit of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase after Expression in Escherichia coli ¹

United States Department of Agriculture, University of Kentucky, Lexington, Kentucky 40546-0076, Agricultural Research Service and Agronomy Department, University of Kentucky, Lexington, Kentucky 40546-0076

The small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) possesses a binding site that can be photoaffinity labeled with [³²P]8-azidoadenosine 5' triphosphate (N₃ATP). In the present study, photoaffinity labeling was used to compare the nucleotide analog binding properties of SSU in the Rubisco holoenzyme complex (holoE SSU) with the properties of isolated SSU and the precursor form (pSSU) that contains a transit peptide. To facilitate these studies, the complete coding regions of tobacco (Nicotiana tabacum L.) SSU and pSSU were cloned into pET expression vectors and the polypeptides were synthesized in Escherichia coli. Protein import studies showed that cloned pSSU polypeptides were imported into intact chloroplasts, where they were processed to the mature form and assembled into the Rubisco holoenzyme. Cloned SSU and pSSU isolated from E. coli were photoaffinity labeled with N₃ATP. The apparent K_D value for SSU and pSSU, 18 micromolar N₃ATP, was identical to the value determined for holoE SSU. However, differences in photolabeling between cloned SSU or pSSU and holoE SSU were apparent in the level of protection afforded by ATP and UTP, in the response of photolabeling to free Mg²⁺, and in the higher photolabeling efficiency that characterized the cloned SSU. Treatment of the Rubisco holoenzyme with a concentration of urea sufficient to disassociate the subunits markedly increased photoincorporation into SSU, indicating that intersubunit associations within the holoenzyme complex may be the major factor influencing photolabeling efficiency of SSU. Thus, differences in SSU conformation between the isolated and assembled states affect photolabeling efficiency and other nucleotide analog binding properties of the SSU, but not the apparent affinity for N₃ATP.

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