Inhibitory effects of nitrite on the reactions of bovine carbonic anhydrase II with CO2 and bicarbonate consistent with zinc-bound nitrite

• Carbonic anhydrase II may generate nitric oxide from nitrite.
• Nitrite inhibits the fast reactions of carbonic anhydrase II with CO2 and bicarbonate.
• The inhibitory effects of nitrite suggest nitrite binding to the active site zinc.
• Carbonic anhydrase II binds its potential physiological substrate nitrite with low affinity.

Carbonic anhydrase (CA) is a zinc enzyme that catalyzes hydration of carbon dioxide (CO2) and dehydration of bicarbonate in red blood cells, thus facilitating CO2 transport and excretion. Bovine CA II may also react with nitrite to generate nitric oxide, although nitrite is a known inhibitor of the CO2 hydration reaction. To address the potential in vivo interference of these reactions and the nature of nitrite binding to the enzyme, we here investigate the inhibitory effect of 10–30 mM nitrite on Michaelis–Menten kinetics of CO2 hydration and bicarbonate dehydration by stopped-flow spectroscopy. Our data show that nitrite significantly affects the apparent dissociation constant KM for CO2 (11 mM) and bicarbonate (221 mM), and the turnover number kcat for the CO2 hydration (1.467 × 106 s− 1) but not for the bicarbonate dehydration (7.927 × 105 s− 1). These effects demonstrate mixed and competitive inhibition for the reaction with CO2 and bicarbonate, respectively, and are consistent with nitrite binding to the active site zinc. The high apparent dissociation constant found here for CO2, bicarbonate and nitrite (16–120 mM) are all overall consistent with published data and reveal a large capacity of free enzyme available for binding each of the three substrates at their in vivo levels, with little or no significant interference among reactions. The low affinity of the enzyme for nitrite suggests that the in vivo interaction between red blood cell CA II and nitrite requires compartmentalization at the anion exchanger protein of the red cell membrane to be physiologically relevant.

Nitrite (NO2−) exerts two types of inhibition of the Michaelis–Menten kinetics of carbonic anhydrase, both consistent with binding to the active Zn2 +: mixed inhibition, with less enzyme available for the CO2 hydration, a process that requires zinc-bound OH−, and competitive inhibition for the bicarbonate dehydration.Figure optionsDownload as PowerPoint slide