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.OH(2), 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(2) 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.OH(2) binding was interpreted as a transformation of the initial "open" intermediate TC.Cbl.OH(2) 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 H(2)O in TC.Cbl.OH(2) for cyanide or azide according to the reaction TC(Cbl) --> TC.Cbl.OH(2) + 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.
Fedosov
S N
SN
Protein Chemistry Laboratory, Department of Molecular and Structural Biology, University of Aarhus, Science Park, Gustav Wieds Vej 10, Denmark. snf@imsb.au.dk
Fedosova
N U
NU
Nexø
E
E
Petersen
T E
TE
D006801:Humans; D006863:Hydrogen-Ion Concentration; D008956:Models, Chemical; D010843:Pichia; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012995:Solubility; D013054:Spectrophotometry, Atomic; D014155:Transcobalamins / Q000737:chemistry* / Q000378:metabolism*; D014805:Vitamin B 12 / Q000031:analogs & derivatives* / Q000378:metabolism
D011994:Recombinant Proteins; D014155:Transcobalamins; C043960:aquacobalamin; D014805:Vitamin B 12