PMID- 8431430
OWN - NLM
STAT- MEDLINE
DCOM- 19930315
LR  - 20190613
IS  - 0006-2960 (Print)
IS  - 0006-2960 (Linking)
VI  - 32
IP  - 6
DP  - 1993 Feb 16
TI  - Engineering the zinc binding site of human carbonic anhydrase II: structure of 
      the His-94-->Cys apoenzyme in a new crystalline form.
PG  - 1510-8
AB  - The structure of the His-94-->Cys variant of human carbonic anhydrase II (CAII) 
      has been determined by X-ray crystallographic methods to a resolution of 2.3 A 
      with a final crystallographic R factor of 0.155. This variant of CAII 
      crystallizes in orthorhombic space group P2(1)2(1)2(1) which is the first example 
      of a new crystal form for this important zinc hydrase (the wild-type enzyme 
      crystallizes in monoclinic space group P21 under similar crystallization 
      conditions). Although the overall structure of the enzyme in the orthorhombic 
      crystal form is similar to that of the wild-type protein in the monoclinic 
      crystal form, the rms deviation of C alpha atoms between the two structures is 
      0.5 A. Larger structural deviations occur in regions of the protein molecule 
      involved in crystal lattice contacts, and significant structural changes are 
      found in the polypeptide strand containing Cys-94. Surprisingly, no electron 
      density corresponding to a zinc ion is found in the active site of crystalline 
      His-94-->Cys CAII, even though the stoichiometry of zinc binding to this variant 
      in solution is confirmed by atomic absorption spectroscopy. However, the KD for 
      zinc dissociation from the variant is increased 10(4)-fold compared with 
      wild-type enzyme; furthermore, under the crystallization conditions of high ionic 
      strength (1.75-2.5 M ammonium sulfate), the observed KD is increased further, 
      which leads to zinc dissociation. Spectroscopic analysis of Co(2+)-substituted 
      His-94-->Cys CAII indicates that the metal binds in a tetrahedral geometry with a 
      new thiolate bond.(ABSTRACT TRUNCATED AT 250 WORDS)
FAU - Alexander, R S
AU  - Alexander RS
AD  - Department of Chemistry, University of Pennsylvania, Philadelphia 19104-6323.
FAU - Kiefer, L L
AU  - Kiefer LL
FAU - Fierke, C A
AU  - Fierke CA
FAU - Christianson, D W
AU  - Christianson DW
LA  - eng
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  - Biochemistry
JT  - Biochemistry
JID - 0370623
RN  - 0 (Apoenzymes)
RN  - 0 (Isoenzymes)
RN  - 0 (Recombinant Proteins)
RN  - 4QD397987E (Histidine)
RN  - EC 4.2.1.1 (Carbonic Anhydrases)
RN  - J41CSQ7QDS (Zinc)
RN  - K848JZ4886 (Cysteine)
SB  - IM
MH  - Amino Acid Sequence
MH  - Apoenzymes/*chemistry/genetics/*metabolism
MH  - Binding Sites
MH  - Carbonic Anhydrases/*chemistry/genetics/*metabolism
MH  - Cloning, Molecular
MH  - *Cysteine
MH  - Escherichia coli/genetics
MH  - *Histidine
MH  - Humans
MH  - Hydrogen Bonding
MH  - Isoenzymes/*chemistry/genetics/*metabolism
MH  - Models, Molecular
MH  - Molecular Sequence Data
MH  - Mutagenesis, Site-Directed
MH  - *Protein Conformation
MH  - Protein Engineering
MH  - Recombinant Proteins/*chemistry/metabolism
MH  - Spectrophotometry
MH  - X-Ray Diffraction
MH  - Zinc/*metabolism
EDAT- 1993/02/16 00:00
MHDA- 1993/02/16 00:01
CRDT- 1993/02/16 00:00
PHST- 1993/02/16 00:00 [pubmed]
PHST- 1993/02/16 00:01 [medline]
PHST- 1993/02/16 00:00 [entrez]
AID - 10.1021/bi00057a015 [doi]
PST - ppublish
SO  - Biochemistry. 1993 Feb 16;32(6):1510-8. doi: 10.1021/bi00057a015.