Functional characterization of human carbonic anhydrase II variants with altered zinc binding sites.
Carbonic anhydrase II (CAII) contains a conserved His3 zinc polyhedron which is essential for catalysis. Removal of any one of the His ligands by replacement with Ala decreases (approximately 10(5)-fold), but does not abolish, zinc binding and increases the rate constant for zinc dissociation. CAII variants with a His ligand substituted with Cys, Asp, or Glu bind zinc only approximately 10-fold better than a His2 zinc polyhedron in CAII. The large decrease in zinc affinity (approximately 5 kcal/mol) in these variants compared to the wild-type His3 site reflects mainly unfavorable compensatory protein structural rearrangements observed in the X-ray crystallographic structures of some of these CAII variants, described by Ippolito and Christianson (following paper in this issue). However, the zinc affinity of these sites is still higher than zinc polyhedra designed de novo. Substitution of the His zinc ligands with negatively charged amino acids both increases the pKa of the zinc-bound water by > or = 1.6 pH units, confirming that neutral ligands maintain the low zinc-water pKa, and decreases the pH-independent kcat/KM for ester hydrolysis (3-30-fold) and CO2 hydration (approximately 10(3)-10(5)-fold). Additionally, decreases in the dissociation constant (approximately approximately 10(2)-10(5)-fold) for the transition state analog acetazolamide correlate with the decreased catalytic efficiency and increased pKa of these CAII variants. These data indicate that the histidine ligands, although not essential for catalysis, are conserved to maximize electrostatic stabilization of both the ground-state zinc-hydroxide and the negatively charged transition state. These studies provide valuable insights into the functional consequences of engineering a catalytic zinc site in a metalloenzyme.
Kiefer
L L
LL
Biochemistry Department, Duke University Medical Center Durham, North Carolina 27710.
Fierke
C A
CA
D001665:Binding Sites; D002245:Carbon Dioxide / Q000737:chemistry; D002256:Carbonic Anhydrases / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008667:Metalloproteins / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D012997:Solvents; D013329:Structure-Activity Relationship; D014867:Water / Q000737:chemistry; D015032:Zinc / Q000737:chemistry
D008667:Metalloproteins; D012997:Solvents; D014867:Water; D002245:Carbon Dioxide; D002256:Carbonic Anhydrases; D015032:Zinc