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Metabolism and Enzymology

Association of Carbonic Anhydrase Activity with Carboxysomes Isolated from the Cyanobacterium Synechococcus PCC7942 ¹

Plant Environmental Biology Group, Research School of Biological Sciences, Australian National University, PO Box 475, Canberra, 2601, Australia, Department of Botany, University of Toronto, 25 Willcocks Street, Toronto, Ontario, Canada M5S 3B2

The development of a simple method for the isolation of purified carboxysomes from the cyanobacterium Synechococcus PCC7942 has made it possible to identify a specific and inducible, intracellular carbonic anhydrase (CA) activity that is strongly associated with carboxysomes. This was shown, in part, through enzyme recovery experiments that indicated that a clear majority of a CA activity that is sensitive to the CA inhibitor ethoxyzolamide (I₅₀ = 4 µM) copurifies with a majority of the cell's ribulose-1,5-bisphosphate carboxylase/oxygenase activity in a highly purified pelletable fraction. Electron microscopy of this pelletable fraction revealed the presence of carboxysomes that were physically intact. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of carboxysome proteins showed that the large and small subunits of ribulose-1,5-bisphosphate carbosylase/oxygenase were clearly prominent and that several other minor proteins could be distinguished. The specific location of this carboxysomal CA activity is further reinforced by the finding that a previously isolated high CO₂-requiring mutant, Type II/No. 68 (G.D. Price, M.R. Badger [1989] Plant Physiol 91: 514-525), displayed a 30-fold reduction in carboxysome-associated CA activity when tested under optimal conditions. Carboxysomal CA has the unusual property of being inactivated by dithiothreitol. The enzyme also requires 20 mM Mg²⁺ (as MgSO₄) for near maximum activity; other divalent cations, such as Ca²⁺ and Mn²⁺, also stimulate carboxysomal CA activity, but to a lesser extent than Mg²⁺. Results are discussed in relation to the role of carboxysomes in the CO₂-concentrating mechanism in cyanobacteria and the role that carboxysomal CA activity appears to play in this process.

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