HUMAN CARBONIC-ANHYDRASE-I - EFFECT OF SPECIFIC SITE MUTATIONS ON ITSFUNCTION

Carbonic anhydrase I (CAI) is one out of ten CA isoenzymes that have b een identified in humans. X-ray crystallographic and inhibitor complex studies of human carbonic anhydrase I (HCAI) and related studies in o ther CA isoenzymes identified several residues, in particular Thr199, Glu106, Tyr7, Glu117, His107, with likely involvement in the catalytic activity of HCAI. To further study the role of these residues, we und ertook, site-directed mutagenesis of HCAI. Using a polymerase chain re action based strategy and altered oligonucleotide primers, we modified a cloned wild type hCAI gene so as to produce mutant genes encoding p roteins with single amino acid substitutions. Thr199Val, Thr199Cys, Th r199Ser, Glu106Ile, Glu106Gln, Tyr7Trp, Glu117Gln, and His107Val mutat ions were thus generated and the activity of each measured by ester hy drolysis. Overproduction of the Glu117Gln and His107Val mutant protein s in Escherichia coil resulted in a large proportion of the enzyme for ming aggregates probably due to folding defect. The mutations Thr199Va l, Glu106Ile and Glu106Gln gave soluble protein with drastically reduc ed enzyme activity, while the Tyr7Trp mutation had only marginal effec t on the activity, thus suggesting important roles for Thr199 and Glu1 06 but not for Tyr7 in the catalytic function of HCAI.