PMID- 9252341
OWN - NLM
STAT- MEDLINE
DCOM- 19970904
LR  - 20210209
IS  - 0021-9258 (Print)
IS  - 0021-9258 (Linking)
VI  - 272
IP  - 33
DP  - 1997 Aug 15
TI  - Selection of carbonic anhydrase variants displayed on phage. Aromatic residues in 
      zinc binding site enhance metal affinity and equilibration kinetics.
PG  - 20364-72
AB  - 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.
FAU - Hunt, J A
AU  - Hunt JA
AD  - Department of Biochemistry, Duke University Medical Center, Durham, North 
      Carolina 27710, USA.
FAU - Fierke, C A
AU  - Fierke CA
LA  - eng
GR  - GM17467/GM/NIGMS NIH HHS/United States
GR  - GM40602/GM/NIGMS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, Non-P.H.S.
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - J Biol Chem
JT  - The Journal of biological chemistry
JID - 2985121R
RN  - EC 4.2.1.1 (Carbonic Anhydrases)
RN  - J41CSQ7QDS (Zinc)
SB  - IM
MH  - Bacteriophages/enzymology
MH  - Binding Sites
MH  - Carbonic Anhydrases/chemistry/*metabolism
MH  - Structure-Activity Relationship
MH  - Zinc/*metabolism
EDAT- 1997/08/15 00:00
MHDA- 1997/08/15 00:01
CRDT- 1997/08/15 00:00
PHST- 1997/08/15 00:00 [pubmed]
PHST- 1997/08/15 00:01 [medline]
PHST- 1997/08/15 00:00 [entrez]
AID - S0021-9258(18)38738-6 [pii]
AID - 10.1074/jbc.272.33.20364 [doi]
PST - ppublish
SO  - J Biol Chem. 1997 Aug 15;272(33):20364-72. doi: 10.1074/jbc.272.33.20364.