A ¹³C Nuclear Magnetic Resonance Study of CO₂/HCO⁻₃ Exchange Catalyzed by Human Carbonic Anhydrase I

Abstract

Rates of COz/HCO; exchange, catalyzed by human carbonic anhydrase I (or B) at chemical equilibrium, were estimated from the nuclear magnetic resonance linewidths of '3C-labeled substrates. The results show that the maximal exchange rate constant is independent of pH in the range 5.7-8.0, whereas the apparent substrate dissociation constant depends on pH. Exchange proceeds rapidly in the absence of added buffers, and the addition of buffers has negligible effects on exchange rates. Exchange is equally rapid with 'HzO or 2Hz0 as solvents. Chloride ions inhibit CO;!/HCO; exchange competitively.The maximal exchange rates obtained with human carbonic anhydrase I are 50 times slower than those obtained with human isoenzyme I1 (or C). From a comparison of the exchange kinetics with the steady-state kinetics of COz hydration and HCOY dehydration it is tentatively concluded that the transfer of H + between active site and medium proceeds with rates of similar magnitudes in the two isoenzynies, whereas the central catalytic step, the interconversion of enzyme-bound COz and HCO;, is much slower in isoenzyme 1 than in isoenzyme 11.Carbonic anhydrase is a zinc-containing metalloenzyme catalyzing the reversible reaction COz + HzO G HCO; + H'.(1) Three isoenzyme types are known to occur in mammals [I]. These have homologous structures indicating common ancestry, and they have different kinetic properties. Carbonic anhydrase I1 (or C) is an extremely efficient catalyst with a CO;! hydration turnover number of lo6 s-' at pH 9 and 25 "C. Carbonic anhydrase I (or B) is less efficient, and the corresponding turnover number is about 10' s-'. Isoenzyme 111 is found in skeletal muscle and has an even lower activity than isoenzyme I. The structural basis of these kinetic differences remains obscure.We have previously performed a series of kinetic studies of human and bovine carbonic anhydrase I1 [2-61. The results can be summarized in a kinetic scheme the main feature of which consists of two half reactions:The interconversion of EH and E-in Eqn (3) is complex involving at least two reaction steps, probably an intramolecular proton transfer [2,7] and a proton transfer between an active-site group and a buffer molecule, which may be regarded as a second substrate [4,8,9]. The rate of exchange between COz and HCOY (Eqn2) can be estimated independently of the proton transfers of Eqn (3) by measuring the enzyme-catalyzed line broadening of 13C NMR resonances of labeled Substrate under equilibrium conditions [lo]. Our previous studies [5] showed that maximal rates of COz/ Enzyme. Carbonic anhydrase (EC 4.2.1.1) HCO; exchange, catalyzed by human carbonic anhydrase 11, are faster that the maximal turnover rates so that turnover must be rate-limited by a step or steps involved in proton transfer between the active site and the medium.We have, then, asked the question if the lower activity of isoenzyme I depends on a slower rate of CO;!/HCOY exchange or a slower rate of proton transfer according to Eqn (3) or both. In this paper we present a ...

13C NMR Characterization of an Exchange Reaction between CO and CO2 Catalyzed by Carbon Monoxide Dehydrogenase

“…If the 13 CO line broadening were to originate from an exchange between 13 CO and 13 CO 2 (or H 13 CO 3 − ) in solution, one would expect to observe a line broadening for both of these resonances, as described in eq 6, which was derived for the 13 CO 2 −H 13 CO 3 exchange catalyzed by carbonic anhydrase (26). Specifically, for the CODH-II reaction, if the CO and CO 2 concentrations are equivalent, the amount of broadening of the 13 CO 2 (or H 13 CO 3 − ) resonance should equal that of the 13 CO resonance.…”

“…The use of this NMR-based 13 CO exchange in the absence of electron carriers also ensures that the chemistry of CO oxidation is followed, not electron transfer, which is rate-limiting under some conditions (19). Assessment of the carbonic anhydrase-catalyzed 13 CO 2 −H 13 CO 3 − exchange reaction provided the inspiration and the theoretical background for this series of experiments (23–26). …”

“…The equilibrium exchange between CO 2 and HCO 3 − , catalyzed by carbonic anhydrase, has been studied by 13 C NMR methods (23–26). This exchange is shown to be pH-independent, in contrast to the sharp pH dependence observed for k cat in the steady-state hydration and dehydration reactions (27).…”

Catalytic and Inhibitor‐Binding Properties of Some Active‐Site Mutants of Human Carbonic Anhydrase I

“…Thus, this mutant does not only show an increased k, , value, but in addition the solvent hydrogen isotope effect in k,,, has increased from 1.7 for wild-type HCA I (Simonsson et al, 1982) to 2.4 for the mutant. This suggests that HCO; dissociation [Eqn (l)], which is thought to dominate over proton transfer [Eqn (2)] as a ratecontrolling step of CO, hydration catalyzed by wild-type HCA I in 1,2-dimethylimidazole buffer (Behravan et al, 1990), contributes less to rate limitation in the mutant.…”

“…The ionic strength was maintained at 0.1 M by the addition of Na,SO,. Data for wild-type isozymes I and I1 are from Simonsson et al (1982) and Simonsson et al (1979), respectively.…”