Human carbonic anhydrase I: Effect of specific site mutations on its function |
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Authors: | A K Mohanty K K Kannan S K Mahajant |
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Institution: | (1) Molecular Biology and Agriculture Division, Bhabha Atomic Research Centre, 400 085 Trombay, Mumbai, India;(2) Solid State Physics Division, Bhabha Atomic Research Centre, 400 085 Trombay, Mumbai, India |
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Abstract: | Carbonic anhydrase I (CAI) is one out of ten CA isoenzymes that have been identified in humans. X-ray crystallographic and
inhibitor complex studies of human carbonic anhydrase I (HCAI) and related studies in other CA isoenzymes identified several
residues, in particular Thr199, GlulO6, Tyr7, Glull7, His l07, with likely involvement in the catalytic activity of HCAI.
To further study the role of these residues, we undertook, site-directed mutagenesis of HCAI. Using a polymerase chain reaction
based strategy and altered oligonucleotide primers, we modified a cloned wild type hCAI gene so as to produce mutant genes
encoding proteins with single amino acid substitutions. Thrl99Val, Thrl99Cys, Thr199Ser, GlulO6Ile, Glul06Gln, Tyr7Trp, Glu.117Gln,
and His 107Val mutations were thus generated and the activity of each measured by ester hydrolysis. Overproduction of the
Glu117Gln and HisI07Val mutant proteins inEscherichia coli resulted in a large proportion of the enzyme forming aggregates probably due to folding defect. The mutations Thr199Val,
GlulO6Ile and GlulO6Gln gave soluble protein with drastically reduced enzyme activity, while the Tyr7Trp mutation had only
marginal effect on the activity, thus s.uggesting important roles for Thr199 and Glu lO6 but not for Tyr7 in the catalytic
function of HCAI. |
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Keywords: | Carbonic anhydrase I site-directed mutagenesis gene expression protein aggregation proton shuttle |
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