In addition to ethanolamine and propanolamine it was found that 2-ethoxyethylamine is a substrate for the corrin-dependent enzyme ethanolamine-ammonia lyase. The enzyme has the same V for 2-ethoxyethylamine as ethanolamine, but the Km for this substrate is 4.3 · 10-3 M. Acetaldehyde and ethylamine were isolated and characterized as reaction products from 2-ethoxyethylamine. When deuterium and tritium replaced hydrogens at the amino carbon of 2-ethoxyethylamine a primary isotope effect was observed for hydrogen transfer with kH k2H = 2.2 and kH k3H = 4.0. Tritium was shown to be transferred from the amino carbon of 2-ethoxyethylamine into the 5′-methylene group of 5,6-dimethylbenzimidazole B12 coenzyme (DMBC) during catalysis. The extent of tritium incorporation into DMBC was shown to be dependent on the ratio of ethanolamine-ammonia tyase holoenzyme to tritiated 2-ethoxyethylamine. Also, tritium was shown to be incorporated into the 5′-methylene group of DMBC from ethanolamine labelled with tritium in the amino carbon, but this exchange was shown to be non-specific. When DMBC was photolysed under anaerobic conditions in the presence of 2-ethoxyethylamine, labelled with tritium in the amino carbon, then the methylene radical generated by photolysis specifically abstracts tritium from the amino carbon of this substrate. The nucleoside product was identified as 5′-deoxyadenosine. When similar photolysis experiements with DMBC were carried out in ethanolamine, tritiated in the amino carbon, non-specific abstraction of both hydrogen and tritium from both carbons was observed. Acetaldehyde was isolated and characterized as a product from both 2-ethoxyethylamine and ethanolamine in these photolysis experiments. The quantity of acetaldehyde formed is directly proportional to the quantity of nucleoside formed by the photolysis of DMBC. These data are consistent with those obtained from enzymatic studies indicating that substrate-dependent homolysis of the CoC σ bond occurs as a prerequisite for hydrogen transfer in ethanolamine-ammonia lyase.
Bibliographical noteFunding Information:
We thank Dr B. M. Babior for his gift of homogeneous ethanolamine-ammonia lyase apoenzyme. This research was supported by a grant from the U.S. Public Health Service AM 12599 .