Kinetic studies of pea carbonic anhydrase.
Chloroplast carbonic anhydrase from Pisum sativum has been isolated. The kinetic properties of the enzyme have been studied and comparisons to the well characterised human carbonic anhydrase II 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 CO2 hydration show an increase in kcat as well as in kcat/Km 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 4 x 10(5) s-1 and a kcat/Km value of 1.8 x 10(8) M-1 s-1 with reference to the subunit. We also find that the enzyme needs high concentrations of buffer to work at a maximal rate. Apparent Km 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 II. Despite those structural differences the kinetic parameters indicate that pea carbonic anhydrase is equally efficient as human carbonic anhydrase II in catalysing the hydration of CO2. However, the mechanism for proton transfer from the active site to the surrounding medium seems to differ between the two enzymes.
Johansson
I M
IM
Department of Biochemistry, University of Umeå, Sweden.
Forsman
C
C
D002256:Carbonic Anhydrases / Q000737:chemistry / Q000378:metabolism*; D002736:Chloroplasts / Q000201:enzymology; D002942:Circular Dichroism; D004789:Enzyme Activation; D004795:Enzyme Stability; D007887:Fabaceae / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010946:Plants, Medicinal; D011487:Protein Conformation
D002256:Carbonic Anhydrases