Conformational changes of transcobalamin induced by aquocobalamin binding - Mechanism of substitution of the cobalt-coordinated group in the bound ligand

Binding of aquo-, cyano-, or azidocobalamin (Cbl . OH2, Cbl . CN, and Cbl . N-3, respectively) to the recombinant human transcobalamin (TC) and haptocorrin from human plasma was investigated via stopped-flow spectroscopy, Association of cobalamins with haptocorrin always proceeded in one step. TC, however, displayed a certain selectivity for the ligands: Cbl . CN or Cbl . N-3 bound in one step with k(+1) = 1 x 10(8) M-1 s(-1) (20 degrees C), whereas binding of Cbl . OH, under the same conditions occurred in two steps with k(+1) = 3 x 10(7) M-1 s(-1) (E-a = 30 kJ/mol) and k(+2) = 0.02 s(-1) (E-a = 120 kJ/mol). The second step of Cbl . OH2 binding was interpreted as a transformation of the initial "open" intermediate TC . Cbl . OH, to the "closed" conformation TC(Cbl) with displaced water. The backward transition from the closed to the open conformation was the reason for the identical rate-limiting steps during substitution of H2O in TC . Cbl . OH2 for cyanide or azide according to the reaction TC(Cbl) --> TC . Cbl . OH2 + CN-/N-3(-). The cyano and azido forms of holo-TC which were produced behaved as the open proteins. Different conformations of holo-TC, determined by the nature of the active group in the bound Cbl, may direct transportation of cobalamins in the organism.