The substrate-dependent steric course of the ethanolamine ammonia-lyase reaction.

Abstract

The adenosylcobalamin‐dependent ethanolamine ammonia‐lyase from Clostridium sp. deaminates (2S)‐2‐aminopropanol to propionaldehyde about three times faster than it does (2R)‐2‐aminopropanol. (2S)‐2‐Amino(1‐2H2)propanol and (1S, 2S)‐2‐amino(1‐2H1)propanol are deaminated by the enzyme with a large kinetic deuterium isotope effect (kH/k2H∼ 6), whereas (1R, 2S)‐2‐amino(1‐2H1)‐propanol behaves like the unlabelled substrate. (2R)‐2‐Amino(1‐2H2)propanol reacts about 1.7 times more slowly than unlabelled (2R)‐2‐aminopropanol or (1R, 2R)‐2‐amino(1‐2H1)propanol. From the above results it follows that the enantiomeric 2‐amino(1‐2H2)propanols react at the same rate, irrespective of their absolute configuration and the lyase reaction involves the 1‐Hsi atom in both enantiomers. (2S)‐2‐Amino[U‐14C, 2‐3H, 3‐3H]propanol was prepared by a combination of enzymic and chemical reactions and the distribution of tritium between C‐2 and C‐3 was determined by its degradation to acetic acid. This doubly labelled substrate was converted on ethanolamine ammonia‐lyase into (2R)‐[U‐14C, 2‐3H, 3‐3H]propionaldehyde. On the other hand, the (2S) configuration was proved for all three samples of [2‐3H]propionaldehyde recovered from incubations of [5′‐3H]adenosylcobalamin with ethanolamine ammonia‐lyase irrespective of whether the (2S) or (2R) enantiomer of 2‐aminopropanol or propionaldehyde itself (in the presence of ammonium ions) were used as substrates. Incubations with enzyme plus [5′‐3H]adenosylcobalamin also yielded tritiated 2‐amino‐propanols. These were in turn incubated with horse liver alcohol dehydrogenase and diaphorase in deuterium oxide, which lead to exchange of the 1‐HRe atom with deuterium. Retention of the tritium in the samples provided evidence for the (1S) configuration. By co‐crystallization as the 4‐methyl‐oxazolidone derivative the (2S) configuration of the tritiated 2‐aminopropanol samples was also proved. These results show that ethanolamine ammonia‐lyase deaminates (2S)‐2‐aminopropanol with retention, whereas (2R)‐2‐aminopropanol reacts with inversion of configuration. Tritium from [5′‐3H]adenosylcobalamin is transferred in all cases to the 2‐pro‐S position of propionaldehyde and to the 1‐pro‐S position of 2‐aminopropanol. In the case of (2R)‐2‐aminopropanol, tritium transfer is accompanied by inversion at the asymmetric centre. All these results, together with the previously reported racemization during the conversion of 2‐aminoethanol, can be explained by a simple mechanistic scheme.