PMID- 8419374
OWN - NLM
STAT- MEDLINE
DCOM- 19930205
LR  - 20210210
IS  - 0021-9258 (Print)
IS  - 0021-9258 (Linking)
VI  - 268
IP  - 2
DP  - 1993 Jan 15
TI  - Determinants of catalytic activity and stability of carbonic anhydrase II as 
      revealed by random mutagenesis.
PG  - 948-54
AB  - The functional importance of a conserved hydrophobic face in human carbonic 
      anhydrase II (CAII), including amino acid residues 190-210, was investigated by 
      random mutagenesis. The catalytic activity, inhibitor binding, and level of CAII 
      expression in Escherichia coli of 57 single amino acid variants were measured 
      revealing that the function of amino acids correlates with their secondary 
      structure placement. Side chains of amino acids in beta-sheet structure are 
      required for the formation of folded, stable protein while those in the turn 
      region determine catalytic efficiency and inhibitor specificity. The CAII active 
      site is extremely plastic, accommodating amino acid substitutions of varied size, 
      charge, and hydrophobicity with little effect on catalysis; only substitutions at 
      Leu198 and Thr199 decrease the rates of CO2 hydration and ester hydrolysis more 
      than 5-fold. These results pinpoint the hydrogen bond network, including the 
      zinc-solvent molecule and Thr199, as crucial for high catalytic efficiency and 
      also suggest that Leu198 forms a portion of a CO2 association site. Increased 
      activity is observed for substitutions at Thr200 (esterase) and Leu203 (hydrase). 
      In addition, the pKa of the zinc-bound water molecule varies upon substitution of 
      amino acids which alter the overall charge of the active site. Three residues 
      interact with sulfonamide inhibitors; substitutions at Thr199 decrease binding 
      (up to 10(3)-fold) while mutations at Thr200 and Cys206 increase binding of 
      dansylamide (up to 80-fold). Mutations in the beta-sheet structure (Asp190-Ser197 
      and Val207-Ile-210) decrease the protein expression of CAII in E. coli, causing 
      the formation of insoluble protein aggregates in many cases. This may suggest an 
      important role for these residues in the folding process. In addition, mutations 
      in Trp192, cis-Pro202, and Trp209 increase thermal lability (up to 5000-fold).
FAU - Krebs, J F
AU  - Krebs JF
AD  - Department of Biochemistry, Duke University Medical Center, Durham, North 
      Carolina 27710.
FAU - Fierke, C A
AU  - Fierke CA
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, P.H.S.
PL  - United States
TA  - J Biol Chem
JT  - The Journal of biological chemistry
JID - 2985121R
RN  - 0 (Dansyl Compounds)
RN  - 0 (Isoenzymes)
RN  - 0 (Recombinant Proteins)
RN  - 1431-39-6 (5-dimethylaminonaphthalene-1-sulfonamide)
RN  - EC 3.1.- (Esterases)
RN  - EC 4.2.1.1 (Carbonic Anhydrases)
RN  - O3FX965V0I (Acetazolamide)
SB  - IM
MH  - Acetazolamide/metabolism
MH  - Amino Acid Sequence
MH  - Base Sequence
MH  - Binding Sites
MH  - Carbonic Anhydrases/chemistry/genetics/*metabolism
MH  - Cloning, Molecular
MH  - Dansyl Compounds/metabolism
MH  - Enzyme Stability
MH  - Escherichia coli/genetics
MH  - Esterases/chemistry/genetics/metabolism
MH  - Humans
MH  - Isoenzymes/chemistry/genetics/*metabolism
MH  - Kinetics
MH  - Models, Molecular
MH  - Molecular Sequence Data
MH  - *Mutagenesis, Site-Directed
MH  - Protein Conformation
MH  - Protein Folding
MH  - Recombinant Proteins/chemistry/metabolism
EDAT- 1993/01/15 00:00
MHDA- 1993/01/15 00:01
CRDT- 1993/01/15 00:00
PHST- 1993/01/15 00:00 [pubmed]
PHST- 1993/01/15 00:01 [medline]
PHST- 1993/01/15 00:00 [entrez]
AID - S0021-9258(18)54025-4 [pii]
PST - ppublish
SO  - J Biol Chem. 1993 Jan 15;268(2):948-54.