Active site and kinetic studies on carbonic anhydrase III.

SHELTON, Jennifer B. (1991). Active site and kinetic studies on carbonic anhydrase III. Doctoral, Sheffield Hallam University (United Kingdom)..

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Carbonic anhydrase III (CA III) was purified to homogeneity from red skeletal muscle of both chicken and human. Analysis of purification procedures revealed that preparations may be easily contaminated with a protein possessing phosphoesterase activity. This could be the source of acid phosphatase activity previously attributed to CA III.The effects of various buffers, anions and phosphorylated metabolites on the activity of these isoenzymes towards bicarbonate and several ester substrates were examined. Phosphate (P[i]) enhanced the HC0[-3] dehydration activity of chicken and human CA III, as measured by the pH-stat assay system. Pipes, mops and hepes buffers had no effect.The K[m] of chicken CA III appeared to decrease with P[i], whereas k[cat] remained constant. Exposure of chicken CA III to high [P[i]] followed by low [P[i]] resulted in retention of P[i]-enhanced activity for up to 20 minutes. This slow dissociation could thus sustain the P[i]-effect under conditions of fluctuating [P[i]]. This response was pH-dependent between pH 6.5-7.5.Pyrophosphate, HSO[-3], ATP, ADP, PEP, 1,3-BPG and 3-PG each enhanced bicarbonate dehydration activity and activation by one species precluded further activation by P[i]. No phosphatase activity by CA III was evident. Activation of CA III by the arginine-modifying reagent, 2,3-butanedione (BD), was also investigated. A comparison of this activation with that of phosphate, for the HC0[-3] dehydration reaction, suggested common features.K[m] and k[cat] were determined for 4-nitrophenyl acetate hydrolysis by chicken CA IE. BD-modification increased k[cat] but had no effect on K[M], whilst P[i] was without effect. This may substantiate the premise that HC0[-3] dehydration and esterase sites are spatially separated on CA IE.The physiological implications of these findings are discussed.

Item Type: Thesis (Doctoral)
Additional Information: Thesis (Ph.D.)--Sheffield Hallam University (United Kingdom), 1991.
Research Institute, Centre or Group - Does NOT include content added after October 2018: Sheffield Hallam Doctoral Theses
Depositing User: EPrints Services
Date Deposited: 10 Apr 2018 17:22
Last Modified: 26 Apr 2021 12:26

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