Some properties of site‐specific mutants of human carbonic anhydrase II having active‐site residues characterizing carbonic anhydrase III

Abstract

Four amino acid residues, His64, Asn67, Leu198 and Val207, in the active site of human carbonic anhydrase II, have been replaced by Lys64, Arg67, Phe198 and Ile207, which are characteristic for the muscle-specific, low-activity isoenzyme form, carbonic anhydrase III. The aim of the investigation has been to test if any of these residues, or a combination of them, is important for the low CO2 hydration activity, low esterase activity, low pKa for the pH/rate profile and low affinity for sulfonamide inhibitors characterizing carbonic anhydrases III. However, no evidence for such critical roles was found. A combination of Lys64 and Arg67 appears to result in a decrease in CO2 hydration activity, but even the quadruple mutant having all four changes is only eight times less active (kcat/Km) than unmodified isoenzyme II, in contrast to isoenzyme III which is nearly 300 times less active than isoenzyme II. The 4-nitrophenyl acetate hydrolase activity of the quadruple mutant is sevenfold lower than that of unmodified isoenzyme II, while the active site of isoenzyme III hardly catalyzes the hydrolysis of this ester at all. The pKa controlling the esterase activity of the quadruple mutant is 6.2, which should be compared to a value of 6.8 for unmodified isoenzyme II, and about 5 for isoenzyme III. While isoenzyme III binds sulfonamide inhibitors 10(3)-10(4) times less strongly than isoenzyme II, only [Asn-67----Arg]isoenzyme II shows a weaker binding of the investigated sulfonamide, dansylamide, but only by a factor of two. Some of the other mutants show enhanced affinities, up to nearly fourfold for the double mutant with Phe198 and Ile207. It is speculated that additional differences between the active sites of isoenzyme II and III might be important for the precise orientations and interactions of the side chains of isoenzyme-III-specific amino acid residues.