Kinetic studies of pea carbonic anhydrase

Abstract

Chloroplast carbonic anhydrase from Pisurn sativurn has been isolated. The kinetic properties of the enzyme have been studied and comparisons to the well characterised human carbonic anhydrase I1 made. Pea carbonic anhydrase was found to be dependent on a reducing agent in order to retain the catalytic activity. Oxidised, inactive, enzyme could be activated by the addition of a SH-agent.However, such activation gave only 60% of the activity of an enzyme kept in a reduced state all the time. The kinetics of CO, hydration show an increase in k,,, as well as in k,,,/K,,, with pH, but the pH profile does not follow a simple titration curve. The pH dependence is more complicated and it seems as if there are several titratable groups affecting the activity. At pH 9 we obtain a turnover number of 4x10' s-' and a k,,,/K,,, value of 1.8X10HM-' s-' with reference to the subunit. We also find that the enzyme needs high concentrations of buffer to work at a maximal rate. Apparent K, values with respect to the total buffer concentration are found between 52-185 mM at neutral and high pH. At low pH the situation is complex with deviations from Michaelis-Menten kinetics.Chloroplast carbonic anhydrase from higher plants have been reported to have primary structures that are completely different from the enzyme from animals. In addition, we find the circular dichroic spectrum of pea carbonic anhydrase to be well distinguished from that of human carbonic anhydrase 11. Despite those structural differences the kinetic parameters indicate that pea carbonic anhydrase is equally efficient as human carbonic anhydrase I1 in catalysing the hydration of CO, However, the mechanism for proton transfer from the active site to the surrounding medium seems to differ between the two enzymes.Carbonic anhydrase (CA; carbonate hydro-lyase) is a zinc-containing enzyme catalysing the reversible hydration of CO,. It is ubiquitous and found in vertebrates, invertebrates, higher plants, algae and in some bacteria. According to the primary structures it seems as if CA has appeared twice during evolution and thus can be divided into two groups. The first group, which is the most studied, contains all so-far-known animal isozymes and the periplasmic CA from Chlarnydomonas reinhardtii. The second group includes chloroplast CA from higher plants together with CA found in two procaryotes. The amino acid sequences for the animal CAs are found to be highly similar with several conserved regions, including the three histidine ligands of the catalytically active zinc ion (Venta et al., 1987). A water molecule is found as the fourth ligand, giving an almost tetrahedral coordination geometry around the zinc ion. In mammals seven different isozymes are known, where most interest has been focused on human CA I and I1 (HCA I and HCA 11) and bovine CA 111. These are monomers with molecular masses around 29 kDa. Their crystal structures have been solved at 0.2-nm resolution or higher and the folding of the polypeptide chain was found to be very similar for all three K...

Structural and Kinetic Characterization of an Archaeal β-Class Carbonic Anhydrase

“…The kinetic properties reported for the P. sativum and Spinacia oleracea (spinach) ␤-class carbonic anhydrases are also consistent with this mechanism (29,30,46). Thus, the kinetic analyses of enzymes from all three classes suggest convergent evolution of the catalytic mechanism (1,36,39).…”

“…The steady-state parameter k cat of the ␤-class S. oleracea carbonic anhydrase was found to be pH dependent, with an apparent pK a of approximately 8.5 in the absence of sulfate (46). Similar to Cab, the pH profiles for both k cat and k cat /K m in the direction of CO 2 hydration for the ␤-class P. sativum carbonic anhydrase are pH dependent, although the profiles were not fitted to theoretical titration curves (29). No significant hydrogen isotope effect was observed on the steady-state parameter k cat /K m for Cab.…”

Identification of a New Chloroplast Carbonic Anhydrase inChlamydomonas reinhardtii

“…by the anions azide and cyanide than bovine CAII, which is an a-CA. Generally, all b-CAs are less sensitive to sulfonamide inhibition than are a-CAs and have sulfonamide I 50 ranging from 2 mM to 10 mM (Johansson and Forsman, 1993). The I 50 of Cah6 falls within this range.…”

Carbonic anhydrases in plants and algae

“…The activity of this enzyme is almost as high as that in the human CAII isoform. However, unlike animal CAs, the plant enzyme is sensitive to oxidation, and reducing conditions in vitro are required to maintain the enzyme in its most active form (Johansson & Forsman 1993;Björkbacka et al 1997). Monocot CAs have not been characterized using physical methods.…”