The proteolytic degradation of normal and variant human carbonic anhydrase isozymes by alpha-chymotrypsin.

The proteolytic degradation by alpha-chymotrypsin of normal and variant human carbonic anhydrase isozymes (CA I and CA II) was investigated by measuring the release of the bound azosulfonamide inhibitor, Neoprontosil, from the active sites of these isozymes. The enzymes studied were the normal isozymes, CA I and CA II, a secondary isozyme of CA I, designated CA I (+1), the CA I variants CA I London (102 Glu leads to Lys), CA I Michigan-2 (100 Thr leads to Lys), and the CA II variant CA II2 (251 Asn leads to Asp). The CA I and CA II isozymes and their respective variants could be classified by their apparent Km values, which were about 40 and 180 microM, respectively. A substrate specificity for alpha-chymotrypsin degradation was given by the ratio kcat/Km, and showed that the specificity decreased such that CA I (+1) > CA I congruent to CA I London congruent to CA I Michigan-2 > CA II2 congruent to CA II. A comparison of the proteolytic data with those previously obtained from thermal inactivation studies of the same isozymes showed agreement in the order of stability to both degradative methods.