Kinetic analysis of a mutant (His107-->Tyr) responsible for human carbonic anhydrase II deficiency syndrome.

The replacement His107-->Tyr is a cause of carbonic anhydrase II deficiency syndrome in humans (Venta, P. J., Welty, R. J., Johnson, T. M., Sly, W. S., and Tashian, R. E. (1991) Am. J. Hum. Genet. 49, 1082-1090). We have prepared this mutant of human carbonic anhydrase II by site-directed mutagenesis and expressed it in Escherichia coli. The mutant was too unstable to purify; however, we were able to stabilize and store it at 4 degrees C in cell lysates containing 1-4 mg/ml bovine serum albumin. The concentration of this mutant in the lysate was determined by titration with the tight-binding inhibitor ethoxzolamide. The stability in this preparation was sufficient to determine that this mutant of carbonic anhydrase II has kcat/Km and apparent pKa for the hydration of CO2 equivalent to that of wild-type HCA II. The maximum velocity of CO2 hydration, which is dependent on the rate of proton transfer between enzyme and solution, was 3-fold smaller than for HCA II suggesting that the proton transfer pathway in the mutant is slightly less efficient than in wild type. Preliminary conformational energy calculations show that the replacement of His107 with the larger residue Tyr results in considerable distortion of the cavity surrounding site 107 and in the loss of at least two hydrogen bonds.