The investigation of cobalt(II) substituted carbonic anhyrase and carboxypeptidase A

Abstract

It is generally accepted that information obtained on cobalt(II) complexes can be transferred to the analogous zinc compounds. Therefore substitution of zinc(II) with cobalt(II) in zinc containing enzymes allows one to investigate these systems through spectroscopic techniques. The data reported here are concerned with carbonic anhydrase (CA) and carboxypeptidase A (CPA). 1H NMR data in D 2O on CoCa reveal a relatively sharp signal assigned to the β proton of the histidine bound to the metal. Its T −1 value is constant with pH for the bovine CA isoenzyme B (BCAB) whereas it s lower at low [H in the case of human CA isoenzyme (HCAB). These data parallel the pH dependence of the water 1H NMR data at every magnetic field [1, 2]. It is proposed that in the latter case (HCAB) a change in coordination chemistry occurs at low pH, and in particular that five coordinate species are obtained, whereas CoBCAB at every pH and CoHCAB at high pH are pseudotetrahedral. The general equilibrium is ▪ Model compounds show that nuclear longitudinal relaxation decreases from tetra- to five- to six-coordination in cobalt(II) complexes. The above equilibrium is consistent with the difference in the electronic spectra and in the pK a between the two isoenzymes. Water 1H NMR data, coordinated histidine 1H NMR data, electronic and EPR data are in our opinion consistent with five-coordination in CoCPA [3]. We believe that five coordination is reached through two water molecules. N 3 − is found to bind ▪ cobalt(II) providing a derivative the electronic spectrum of which can be interpreted as being due to tetracoordination. The affinity of N 3 − for the enzyme decreases with pH, the pK a being around 9. This behavior of N 3 − is analogous to that shown with CoHCAB both with respect to change in coordination number and pH dependence of the affinity constants.