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Human carbonic anhydrase I: Effect of specific site mutations on its function

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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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Abbreviations

For genes :

hCAI

Human carbonic anhydrase I:

ehCAI

erythrocyte human carbonic anhydrase I:

rhCAI

recombinant human carbonic anhydrase I. For proteins:

CAI

Carbonic anhydrase I:

eHCAI

erythrocyte human carbonic anhydrase I:

rHCAI, recombinant human carbonic anhydrase I

HCAI:

human carbonic anhydrase I

HCAII:

human carbonic anhydrase II

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Authors and Affiliations

  1. Molecular Biology and Agriculture Division, Bhabha Atomic Research Centre, 400 085, Trombay, Mumbai, India

    A. K. Mohanty & S. K. Mahajant

  2. Solid State Physics Division, Bhabha Atomic Research Centre, 400 085, Trombay, Mumbai, India

    A. K. Mohanty & K. K. Kannan

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  1. A. K. Mohanty
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  2. K. K. Kannan
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  3. S. K. Mahajant
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Correspondence to S. K. Mahajant.

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Mohanty, A.K., Kannan, K.K. & Mahajant, S.K. Human carbonic anhydrase I: Effect of specific site mutations on its function. J. Biosci. 23, 235–246 (1998). https://doi.org/10.1007/BF02720025

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