Kinetic characterization of wild-type and proton transfer-impaired variants of beta-carbonic anhydrase from Arabidopsis thaliana.
We have cloned and overexpressed a truncated, recombinant form of beta-carbonic anhydrase from Arabidopsis thaliana. The wild-type enzyme and two site-directed variants, H216N and Y212F, have been kinetically characterized both at steady state by stopped-flow spectrophotometry and at chemical equilibrium by (18)O isotope exchange methods. The wild-type enzyme has a maximal k(cat) for CO2 hydration of 320 ms(-1) and is rate limited by proton transfer involving two residues with apparent pK(a) values of 6.0 and 8.7. The mutant enzyme H216N has a maximal k(cat) at high pH that is 43% that of wild type, but is only 5% that of wild type at pH 7.0. (18)O exchange studies reveal that the effect of the mutations H216N or Y212F is primarily on proton transfer steps in the catalytic mechanism and not in the rate of CO2-HCO3- exchange. These results suggest that residues His-216 and Tyr-212 are both important for efficient proton transfer in A. thaliana carbonic anhydrase.
Rowlett
Roger S
RS
Department of Chemistry, Colgate University, Hamilton, NY 13346, USA. rrowlett@mail.colgate.edu
Tu
Chingkuang
C
McKay
Melissa M
MM
Preiss
Jeffrey R
JR
Loomis
Rebecca J
RJ
Hicks
Katherine A
KA
Marchione
Robb J
RJ
Strong
Jacob A
JA
Donovan
George S
GS
Chamberlin
Joy E
JE
D019943:Amino Acid Substitution; D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics; D001665:Binding Sites / Q000502:physiology; D002245:Carbon Dioxide / Q000737:chemistry; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000235:genetics*; D002384:Catalysis; D004789:Enzyme Activation / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000737:chemistry; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010103:Oxygen Isotopes; D011522:Protons; D013379:Substrate Specificity; D015032:Zinc / Q000032:analysis
D029681:Arabidopsis Proteins; D007093:Imidazoles; D007527:Isoenzymes; D010103:Oxygen Isotopes; D011522:Protons; D002245:Carbon Dioxide; C029899:imidazole; D002256:Carbonic Anhydrases; D015032:Zinc