Possible roles for His 208 in the active-site region of chloroplast carbonic anhydrase from Pisum sativum.
His 208 of chloroplast pea carbonic anhydrase is conserved among the dicotyledonous carbonic anhydrases. This His was replaced by an Ala to test whether a histidine residue at this position, in analogy to His 64 of human carbonic anhydrase II, acts as an internal proton shuttle. Values of the kinetic parameters kcat and kcat/Km for the H208A mutant enzyme are high over the pH range 6-9 and of the same magnitude as those for the wild-type enzyme, indicating that this residue is not crucial for the catalytic event. The pH dependence of kcat/Km, reflecting the Zn-H2O ionization, is, however, simplified to that of a simple titration with pKa = 7.1 +/- 0.1 in the absence of His 208. Interaction with the proton-accepting buffer molecule is impaired in the mutant, and apparent Km values for the buffer have increased up to 20 times. Furthermore, the H208A mutant is more easily inactivated by oxidation than the wild-type enzyme. The results indicate that the pKa for a redox-sensitive Cys residue is decreased by at least one pH unit in the mutant, and the histidyl side chain seems to have a function in stabilizing the reduced and active form of the enzyme. An interaction with the redox-sensitive cysteines at positions 269 and 272 is proposed.
Björkbacka
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H
Department of Biochemistry, Umeâ University, S-901 87, Umeâ, Sweden.
Johansson
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IM
Forsman
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C
D001665:Binding Sites / Q000235:genetics; D002257:Carbonic Anhydrase Inhibitors / Q000494:pharmacology; D002256:Carbonic Anhydrases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002736:Chloroplasts / Q000201:enzymology* / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D006639:Histidine / Q000235:genetics / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D018532:Pisum sativum / Q000201:enzymology*
D002257:Carbonic Anhydrase Inhibitors; D006639:Histidine; D002256:Carbonic Anhydrases