Interaction of lead ions with bovine carbonic anhydrase: further studies

Abstract

Lead-substituted bovine carbonic anhydrase is investigated and the return to the holoenzyme form with exchange of Pb 2+ by Zn 2+ is followed by uv difference spectroscopy and by esterase activity methods. Equimolar amounts of Pb 2+ added to apocarbonic anhydrase release one hydronium ion per molecule below pH 6. Above this pH there is a net gain of hydronium ions by the enzyme, due to Pb(OH) + → Pb(OH 2) 2 +, when the metal is bound within the active site of the enzyme molecule. The reduced hydrolysis by lead when it is bound to the enzyme is relevant to the theory of Zn 2+ hydrolysis as a mechanism for carbon dioxide hydration by the holoenzyme and to the idea of an altered p K hydrolysis when Zn 2+ is bound in the enzyme active site cavity. Lead appears to be bound to a His residue in the active site and to interact with a Tyr residue nearby. The Tyr interaction is disrupted by a high concentration of chloride ions, (also by lower concentrations of cyanide ions), but such anions do not displace lead from the enzyme. At pH 8.0 the buffer-free exchange of Pb 2+ by Zn 2+ is found to be consistent with a second-order process with an effective β = (95 ± 7) M −1 sec −1. Thus lead is more rapidly replaced by zinc than is Mn 2+ or VO 2+ whose replacement kinetics have been reported by others. Comparison of esterase-activation and spectral curves with second-order models shows that the effective β is both large and buffer dependent, indicating that a proton transfer process or buffer anion effects may be rate limiting in the buffer-free case.