VANADATE-DEPENDENT BROMO IODOPEROXIDASE FROM ASCOPHYLLUM NODOSUM ALSO CONTAINS UNSPECIFIC LOW-AFFINITY BINDING-SITES FOR VANADATE(V) - A V-51 NMR INVESTIGATION, INCLUDING THE MODEL PEPTIDES PHE-GLU AND GLY-TYR

The interaction of vanadate(V) with bromo/iodoperoxidase from the cell wall of knobbed wrack (Ascophyllum nodosum), A.n.I, has been investigated by V-51 NMR. With excess vanadate, the holoenzyme (E*) forms unspecific associates. The main component is a complex (VE*)A with K(A) = 16(7) M-1 (250(25) M-1 in the presence of KBr, pH = 7.3)) and a chemical shift delta-(V-51) = -498 to -512 ppm relative to VOCI3. The enhancement of binding by substrate bromide is discussed in terms of a stepwise incorporation of vanadate into the active site of the enzyme. Additional, very weak complexes have been detected, among these a tetrahedral one (possibly hydrogen bonded monovanadate) in exchange with free monovanadate at medium exchange rates. Characteristics similar to those for (VE*)A have been observed for complexes of vanadate with the dipeptides Phe-Glu and Gly-Tyr, suggesting similar coordination modes. Phe-Glu, at pH 7.5, forms a mono- and dinuclear, monoligate complex (delta = -515 ppm; K for the mononuclear species is 322 M-1). The complexes obtained from the interaction between vanadate and Gly-Tyr (K = 128 M-1, delta = - 510 at pH = 7.2; delta = - 500 and - 506 ppm at pH = 8.2) are mononuclear and monoligate. Tentative formulations for the coordination compounds consider side chain functions, the peptide linkage, and the terminal amino group.