The Mechanism of Action of Ethanolamine Deaminase: IV. Cobamide-dependent binding of substrate to ethanolamine deaminase

Abstract

Abstract In the presence of a number of alkyl cobalamins, a complex is formed between ethanolamine deaminase and radioactive ethanolamine which can be isolated by gel filtration. Studies with the methylcobalamin-dependent complex have shown that the amount of substrate bound reaches a maximum at a cobamide to enzyme ratio of 2. The binding of substrate to this complex is depressed by ethanolamine analogues which are competitive inhibitors of the catalytic reaction. Measurements of binding as a function of substrate concentration indicate a dissociation constant for the complex of 2 x 10-7 m, a value which is surprisingly high in view of the apparent stability of the complex. Incubating the complex with unlabeled ethanolamine or allowing it to reside on the gel column for various periods of time leads to a loss of radioactivity from the complex which is almost complete by 6 min. This loss does not occur if the complex is exposed to light prior to incubation with unlabeled substrate. However, exposure of a mixture of enzyme and methylcobalamin to light in the absence of substrate prevents the complex from forming when radioactive substrate is added. Although the photolyzed complex is stable in the presence of unlabeled substrate, treatment with 6 m urea results in the release of all the radioactivity from the complex. With both the unphotolyzed and the photolyzed complexes, the radioactive compound associated with the enzyme was shown to be unchanged ethanolamine. These results are interpreted as suggesting that the formation of the enzyme-substrate complex is reversible in practical terms, but that in the presence of certain cobalamin derivatives the dissociation of the complex is slowed to the extent that it can be observed experimentally. Thus, the apparent stability of the complex is kinetic rather than thermodynamic in origin.