Enzymatic Iodination of Protein

• Published in: European journal of biochemistry Vol. 38; no. 3; pp. 497 - 506
• Main Authors: Pommier, Jacques, Sokoloff, Louis, Nunez, Jacques
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.10.1973
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1111/j.1432-1033.1973.tb03085.x

1. The initial rate of I2 formation catalyzed by horse‐radish peroxidase exhibited a clear sigmoid relationship with respect to the concentration of I−. These data were found to fit an equation based on a model which predicts a second‐order dependence on iodide concentration and are consistent with a bimolecular reaction between two I−ions on the surface of the enzyme. Such a model presumes two sites for the substrate on the enzyme. 2. The initial rate of lysozyme iodination also exhibited a clear sigmoid relationship with respect to concentration of I−. The sigmoidicity increased with the lysozyme concentration. These experimental data fit a random‐ordered sequence of substrate fixation but with one of the two possible sequences kinetically preferred. These results also suggest that lysozyme interacts with the enzyme and that there are two sites for substrate addition on the surface of the enzyme. 3. When the ratio of the concentrations of both substrates, iodide and lysozyme, is varied, the rates of formation of each product, I2 or iodinated protein, also vary; the lower the iodide/protein ratio, the lower the I2 yield and the higher the rate of protein iodination. Studies on the influence of pH on the nature of the product showed that I2 formation is favored at acidic pH and protein iodination at more alkaline pH. These results are also consistent with a two‐site model for the enzyme, both sites being able to fix either two iodide ions or one iodide ion and one lysozyme molecule. The affinity of the sites for each one of the two substrates differs according to the pH. This conclusion was confirmed by the observation that iodination of free tyrosine could be obtained with very good yields provided that the pH of the reaction was adjusted to avoid either complete dimerization of free tyrosine, which is favored at alkaline pH, or I2 formation which is favored at low pH. 4. These results and other quantitative data on the stoichiometry of H2O2 consumption do not establish unequivocally which is the oxidized iodide‐reacting species, I+ or I°. However, they indicate that I2 is not the iodinating species in the protein iodination reaction catalyzed by horse‐radish peroxidase.