Kinetic pathways and carbonic anhydrase mechanisms

Carbonic anhydrase (CA) exists in three forms: the low-pH form (L); the high-pH form (H); and the anion-inhibited from (A). The latter includes the bicarbonate complex. All three forms have been demonstrated in CA I and, when sulfate is removed, in CA II. The L-form of CA III has not yet been seen, even at pH 5. Equilibrium among the three forms in a sample of CA can be established, in principle, by kinetic pathways connecting any two forms; which pathway dominates is as yet an open question. By invoking the usual ping-pong mechanism of CA, during which hydration of CO₂ causes the enzyme to go from H to L, the kinetic pathway connecting A and H is ignored, essentially by definition. Rarely has the AH pathway been considered (cf. Koenig et al., 1980). Though there are few data to demonstrate the relative kinetics of the AL and AH pathways, it can be argued that the latter is buffer-mediated, which could distinguish the two. In this case, the lifetime of a bound anion would be buffer-dependent. We have investigated this point by measuring the nuclear relaxation rates of fluorine of trifluoroacetate in Co²⁺CA II solutions. The fluorine linewidth, and thus the anion exchange rate, is independent of buffer concentration up to ～50 mM, which argues for the AL pathway predominating.