Biological Oxidation-Elimination-Addition and Related Sequences

8 BIOLOGICAL OXIDATION-ELIMINATION-ADDITION AND RELATED SEQUENCES 

X-ray crystallography of the recombinant rat liver and human enzyme have fleshed out some details of Palmer-Abeles mechanism. The wt enzyme crystallises in an open conformation, which on binding of 

The stereochemistries of the reactions in the adenosyl moiety were determined by H NMR spectroscopy of the H5 resonances of C5 chirally deuterated substrates. First, the C5 stereochemistries of the 5 -adenosylho-mocysteine produced from chirally deuterated adenosine and homocysteine were compared with samples of known stereochemistry the overall reaction proceeded with retention of configuration. Then the stereochemistry of the addition reaction was examined by examining the H5 resonances of the homocysteine produced from ( )- and (Z)-5 -deutero-4,5-dehydro-3-ketoade-nosine the stereochemistry of addition was syn. Therefore, because of the overall retention of configuration, the stereochemistry of both elimination and addition is syn. 

Pre-steady-state kinetic studies established that the appearance of the NADH chromophore on addition of substrate was a two-step process, and these steps can now be identified as closure of the active site and hydride transfer. This study indicated that the on-enzyme equilibrium for addition of water or homocysteine to the enone was close to unity (and the value in free solution), whereas the equilibrium for oxidation of NAD by bound adenosine was 10 times more favourable than in free solution. The focusing of the catalytic power of the enzyme on the oxidation step avoids the formation of abortive complexes by hydride transfer between enone and NADH, yielding 4,5-dehydroadenosine and NAD . This happens about 10 times faster than productive hydride transfer at the beginning and end of the catalytic cycle, with the slow rate (close to that of model reactions) apparently arising from a conformationally modulated increase in the distance the hydride has to be transferred. 

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