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.