Linked Life Data

9585580

Source:http://linkedlifedata.com/resource/pubmed/id/9585580

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
20
pubmed:dateCreated
1998-6-22
pubmed:abstractText
The hydration of CO2 catalyzed by carbonic anhydrase requires proton transfer from the zinc-bound water at the active site to solution for each cycle of catalysis. In the most efficient of the mammalian carbonic anhydrases, isozyme II, this transfer is facilitated by a proton shuttle residue, His 64. Murine carbonic anhydrase V (mCA V) has a sterically constrained tyrosine at the analogous position; it is not an effective proton shuttle, yet catalysis by this isozyme still achieves a maximal turnover in CO2 hydration of 3 x 10(5) s-1 at pH > 9. We have investigated the source of proton transfer in a truncated form of mCA V and identified several basic residues, including Lys 91 and Tyr 131, located near the mouth of the active-site cavity that contribute to proton transfer. Intramolecular proton-transfer rates between these shuttle groups and the zinc-bound water were estimated as the rate-determining step in kcat for hydration of CO2 measured by stopped-flow spectrophotometry and in the exchange of 18O between CO2 and water measured by mass spectrometry. Comparison of kcat in catalysis by Lys 91 and Tyr 131 and the corresponding double mutant showed a strong antagonistic interaction between these sites, suggesting a cooperative behavior in facilitating the proton-transfer step of catalysis. Replacing four potential proton shuttle residues produced a multiple mutant that had 10% of the catalytic turnover kcat of the wild type, suggesting that the main proton shuttles have been accounted for in mCA V. These replacements caused relatively small changes in kcat/Km for hydration, which measures the interconversion of CO2 and HCO3- in a stage of catalysis that is separate and distinct from the proton transfers; these measurements serve as a control indicating that the replacements of proton shuttles have not caused structural changes that affect reactivity at the zinc.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
May
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
19
pubmed:volume
37
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
7649-55
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1998
pubmed:articleTitle
Intramolecular proton transfer from multiple sites in catalysis by murine carbonic anhydrase V.
pubmed:affiliation
Department of Biochemistry, University of Florida College of Medicine, Gainesville 32610-0267, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.