Measuring carbonic anhydrase activity in alpha-carboxysomes using stopped-flow.

Carboxysomes are protein-based organelles that serve as the centerpiece of the bacterial CO₂ concentration mechanism (CCM). They are present in all cyanobacteria and many chemoautotrophic proteobacteria and encapsulate the key enzymes for CO₂ fixation, carbonic anhydrase and the carboxylase Rubisco, within a protein shell. The CCM actively accumulates bicarbonate in the cytosol, which diffuses into the carboxysome where carbonic anhydrase rapidly equilibrates it to CO₂. This creates a high CO₂ concentration around Rubisco, ensuring efficient carboxylation. In this chapter, we present a general method for purifying α-carboxysomes and measuring carbonic anhydrase activity within these purified compartments. We exemplify this with α-carboxysomes purified from the chemoautotroph Halothiobacillus neapolitanus c2, a model organism for the α-carboxysome based CCM. However, this purification protocol can be adapted for other species, such as carboxysomes from α-cyanobacteria or carboxysomes expressed in heterologous hosts. Further, we describe the Khalifah/pH indicator assay for measuring steady-state kinetics of carbonic anhydrase catalyzed CO₂ hydration. This method allows us to determine the kinetic parameters k_cat, K_M and k_cat/K_M for the purified α-carboxysomes. It uses a stopped-flow spectrometer for rapid mixing and detection, crucial for capturing the fast equilibrium between CO₂ and bicarbonate. The reaction progress is monitored by absorbance via a pH indicator that changes color due to the proton release. While the method specifically focuses on measuring carbonic anhydrase activity on carboxysomes, it can be used to measure activity on carbonic anhydrases from other contexts as well.