Linked Life Data

7925414

Source:http://linkedlifedata.com/resource/pubmed/id/7925414

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
3
pubmed:dateCreated
1994-10-27
pubmed:abstractText
Chloroplast carbonic anhydrase from Pisum sativum has been studied to elucidate the catalytic mechanism and to test if the mechanism proposed for human carbonic anhydrase II is also valid for pea carbonic anhydrase. The catalytic activity was found to depend on the chemical nature of the buffer. Barbital buffer gives the highest turnover number at infinite buffer concentration and the lowest Km value with respect to the buffer, while the kinetic parameters obtained in the imidazole-type buffer, 1-methylimidazole, do not differ from those obtained using the biological-type buffer Mops. The anion inhibition of CO2 hydration was investigated using SCN- at pH 6-9. The binding of the anion was found to be pH dependent with the strongest interaction at low pH. We obtained an uncompetitive inhibition pattern at high pH and noncompetitive inhibition patterns at pH 7 and low pH. The catalytic mechanism was further tested by measurements of the solvent hydrogen isotope effects on the kinetic parameters for CO2 hydration. The observed effects were comparatively small with a kcat value of approximately 2 irrespective of the pH. The effect on kcat/Km and on Km changes when going from high pH to pH 7 and low pH. At high pH, the solvent isotope effect in Km is at least 3, giving a value below 1 for kcat/Km, while at pH 7 and low pH the major effect is found in kcat/Km with values of 2.6 and 2.9. The dependence of the CO2-hydration activity on the buffer concentration is in agreement with a ping-pong mechanism with buffer acting as a second substrate. This is analogous to the behaviour of human carbonic anhydrase II. The inhibition patterns and the observed isotope effects at high pH can also be explained within the framework of the catalytic mechanism for human carbonic anhydrase II, with a rate-determining and buffer-dependent part. The results are consistent with a mechanism involving a proton transfer that contributes to rate limitation. However, the isotope effects found at pH 7 and low pH indicate that some part of the mechanism has changed. Moreover, we cannot decide whether the mechanism for pea carbonic anhydrase involves an internal proton-shuttle group, or if the buffer molecule acts in a direct proton transfer from the zinc-coordinated water.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Sep
pubmed:issn
0014-2956
pubmed:author
pubmed:issnType
Print
pubmed:day
15
pubmed:volume
224
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
901-7
pubmed:dateRevised
2007-7-23
pubmed:meshHeading
pubmed:year
1994
pubmed:articleTitle
Solvent hydrogen isotope effects and anion inhibition of CO2 hydration catalysed by carbonic anhydrase from Pisum sativum.
pubmed:affiliation
Department of Biochemistry, University of Umeå, Sweden.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't