PMID- 7803386
OWN - NLM
STAT- MEDLINE
DCOM- 19950127
LR  - 20141120
IS  - 0006-2960 (Print)
IS  - 0006-2960 (Linking)
VI  - 33
IP  - 51
DP  - 1994 Dec 27
TI  - Structural consequences of redesigning a protein-zinc binding site.
PG  - 15241-9
AB  - In order to probe the structural importance of zinc ligands in the active site of 
      human carbonic anhydrase II (CAII), we have determined the three-dimensional 
      structures of H94C (in metal-bound form), H94C-BME (i.e., disulfide-linked with 
      beta-mercaptoethanol), H94A, H96C, H119C, and H119D variants of CAII by X-ray 
      crystallographic methods at resolutions of 2.2, 2.35, 2.25, 2.3, 2.2, and 2.25 A, 
      respectively. Each variant crystallizes isomorphously with the wild-type enzyme, 
      in which zinc is tetrahedrally coordinated by H94, H96, H119, and hydroxide ion. 
      The structure of H94C CAII reveals the successful substitution of the naturally 
      occurring histidine zinc ligand by a cysteine thiolate, and metal coordination by 
      C94 is facilitated by the plastic structural response of the beta-sheet 
      superstructure. Importantly, the resulting structure represents the catalytically 
      active form of the enzyme reported previously [Alexander, R. S., Kiefer, L. L., 
      Fierke, C. A., & Christianson, D. W. (1993) Biochemistry 32, 1510-1518]. 
      Contrastingly, the structure of H96C CAII reveals that the engineered side chain 
      does not coordinate to zinc; instead, zinc is tetrahedrally liganded by H94, 
      H119, and two solvent molecules. Thus, the beta-sheet superstructure is not 
      sufficiently plastic in this location to allow C96 to coordinate to the metal 
      ion. Substitution of the thiolate or carboxylate group for wild-type histidine in 
      H119C and H119D CAIIs reveals that tetrahedral metal coordination is maintained 
      in each variant; however, since there is no plastic structural response of the 
      corresponding beta-strand, a longer metal-ligand separation results.(ABSTRACT 
      TRUNCATED AT 250 WORDS)
FAU - Ippolito, J A
AU  - Ippolito JA
AD  - Department of Chemistry, University of Pennsylvania, Philadelphia 19104-6323.
FAU - Christianson, D W
AU  - Christianson DW
LA  - eng
GR  - GM08275/GM/NIGMS NIH HHS/United States
PT  - Journal Article
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  - Biochemistry
JT  - Biochemistry
JID - 0370623
RN  - 0 (Metalloproteins)
RN  - 0 (Recombinant Proteins)
RN  - EC 4.2.1.1 (Carbonic Anhydrases)
RN  - J41CSQ7QDS (Zinc)
SB  - IM
MH  - Binding Sites
MH  - Carbonic Anhydrases/*chemistry
MH  - Computer Graphics
MH  - Crystallography, X-Ray
MH  - Fourier Analysis
MH  - Humans
MH  - In Vitro Techniques
MH  - Metalloproteins/*chemistry
MH  - Protein Structure, Tertiary
MH  - Recombinant Proteins
MH  - Structure-Activity Relationship
MH  - Zinc/*chemistry
EDAT- 1994/12/27 00:00
MHDA- 1994/12/27 00:01
CRDT- 1994/12/27 00:00
PHST- 1994/12/27 00:00 [pubmed]
PHST- 1994/12/27 00:01 [medline]
PHST- 1994/12/27 00:00 [entrez]
PST - ppublish
SO  - Biochemistry. 1994 Dec 27;33(51):15241-9.