Phytic acid-enhanced metal ion exchange reactions: the effect on carboxypeptidase A.

On the basis of the known interaction of phytic acid to form soluble or insoluble complexes with cations, the effect of this naturally occurring polydentate ligand on carboxypeptidase A, a zinc-containing metalloenzyme, and its Co(II)-substituted derivative, has been studied. Under conditions of rigorous exclusion of adventitious metal ions, phytate showed no inhibitory effect. However, the addition of Cu(II) ions to form soluble phytate-Cud(II) complexes at pH 7.2 and 25° C caused more than a 95% decrease in activity. The Cd(II) ion was nearly as effective but other ions showed only a small or no effect. In the absence of phytate, incubation of the enzyme with Cu(II) or Cd(II) at the same concentration produced only about a 25% reduction in activity. The decrease in activity followed first-order kinetics, and the rate constant was the same (1.2 × 10−4 sec−1) as seen upon incubation with EDTA. However, in contrast to that observed upon incubation of the enzyme with phytate and Cu(II), exposure to EDTA produced a complete loss in activity which could be regained by addition of Zn(II) to the assay solution. In the former case, not only was there residual activity left after incubation at pH 7.2 for 24 hrs at 25°C, but the initial activity could not be regained under similar assay treatment. An increase in either the Cu(II) or phytate concentration while the other was kept constant, yielded saturation curves with maximal effect at 3 × 10−5 M for Cu(II) and at 5 × 10−5 M for phytate (enzyme at ca. 10−6 M). At these ratios, all of the cupric ions are completely bound to phytate as determined by ion-selective potentiometry. A preparative scale reaction of phytate and Cu(II) with carboxypeptidase A (kcat 8460 min−1 KM′0.23 mM with CBZ-glycyl-glycyl-L-phenylalanine as substrate at pH 7.5, 25°C) gave a product isolated in 95% yield but with lower activity (kcat 198 min−1; KM′0.25 mM). A Cu(II)-carboxypeptidase preparation had similar kinetic parameters (kcat 207 min−1; KM′0.34 mM). This near identity of constants suggested that a metal exchange reaction had occurred, i.e., incubation of Zn(II)-carboxypeptidase with a phytate-Cu(II) complex resulted in not only the removal of the zinc ion from the active site but also the sequential and rapid incorporation of a cupric ion into the apoenzyme so formed. The independently prepared Cu(II)-substituted enzyme preparation and the product of the reaction between carboxypeptidase A and phytate with Cu(II) could be completely converted back to the original enzyme by exposure to an overwhelming Zn(II) concentration. These results provide evidence for the chemical potential of phytate in combination with Cu(II) ions to cause a marked reduction in the activity of carboxypeptidase A through the mechanism of a cation exchange reaction that results in the formation of a copper-substituted enzyme. Since this, in a kinetic sense, is equivalent to inhibition, it adds a new dimension to the nutritional significance of phytate. In addition to phytate being considered the culprit in foodstuffs that adversely impacts the bioavailability of metal ions, the possibility exists that phytate may also interfere with the terminal digestion stage of proteins.