Selection of carbonic anhydrase variants displayed on phage. Aromatic residues in zinc binding site enhance metal affinity and equilibration kinetics.
In all metalloenzymes, hydrophobic residues surround the metal binding site. In carbonic anhydrase II (CAII) residues Phe93, Phe95, and Trp97 flank two of the three histidines that coordinate zinc to form a hydrophobic cluster beneath the zinc binding site. A library of CAII variants differing in these hydrophobic amino acids was prepared using cassette mutagenesis, then displayed on filamentous phage, and screened for proteins retaining high zinc affinity. Wild-type CAII was enriched 20-fold by selection, and consensus residues at each position were identified from the enriched CAII variants (Ile, Phe, Leu, and Met at position 93; Ile, Leu, and Met at position 95; and Trp and Val at position 97). Highly selected variants have zinc affinity and catalytic activity nearly equal to that of wild-type CAII, indicating that the aromatic residues are not absolutely essential. However, the zinc dissociation rate constant and catalytic activity of the variants correlate with the volume of the amino acids at positions 93, 95, and 97. In summary, metalloenzyme variants displayed on phage can be selected on the basis of metal affinity; such methods will be useful for optimization of metal ion biosensors.
Hunt
J A
JA
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Fierke
C A
CA
D001435:Bacteriophages / Q000201:enzymology; D001665:Binding Sites; D002256:Carbonic Anhydrases / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship; D015032:Zinc / Q000378:metabolism*
D002256:Carbonic Anhydrases; D015032:Zinc