Carbonic anhydrase IV (CA IV) is a membrane-bound form of carbonic anhydrase. We have characterized the catalytic activity and inhibition of recombinant human CA IV. CA IV is a high-activity isozyme in CO2 hydration with a pH-independent kcat value (1.1 x 10(6) s(-1)) comparable to that of CA II (8 x 10(5) s(-1)). Furthermore, CA IV is more active in HCO3- dehydration than is CA II as illustrated by the nearly 3-fold increase in kcat/K(M) to 3 x 10(7) M(-1) s(-1). However, the esterase activity of CA IV is decreased 150-fold compared to CA II. The catalytic mechanisms of CA II and CA IV are nearly identical. Both isozymes show similar dependence on buffer concentration with the rate-limiting step at high buffer concentration being intramolecular proton transfer, although the intramolecular proton transfer for CA IV is 3 times faster than that observed with CA II. Additional positive charges in the active site of CA IV stabilize anions as indicated by a decreased pKa for the Zn-bound water compared to CA II (6.2 vs 6.9), as well as lower inhibition constants for a variety of anions, including halides, sulfate, formate, acetate, and bicarbonate. CA IV is also activated by low concentrations (<20 mM) of chloride, bromide, and phosphate. Activation by phosphate suggests that the phospholipid anchor may be acting both as an extracellular tether and as a protein activator. Finally, the affinity of CA IV for sulfonamide inhibitors is decreased up to 65-fold compared to CA II as demonstrated by fluorescence titration. The increased bicarbonate activity and altered pH profile are consistent with the proposed physiological role of CA IV in renal bicarbonate reabsorption.