N-acylglycine amidation: Implications for the biosynthesis of fatty acid primary amides

Bifunctional peptidylglycine alpha-amidating enzyme (alpha-AE) catalyzes th e O-2-dependent conversion of C-terminal glycine-extended prohormones to th e active, C-terrninal cl-amidated peptide and glyoxylate. We show that alph a-AE will also catalyze the oxidative cleavage of N-acylglycines, from N-fo rmylglycine to N-arachidonoylglycine. N-Formylglycine is the smallest amide substrate yet reported for alpha-AE. The (V/K)(app) for N-acylglycine amid ation varies similar to 1000-fold, with the (V/K)(app) increasing as the ac yl chain length increases. This effect is largely an effect on the K-M,K-ap p; the K-M,K-app for N-formylglycine is 23 +/- 0.88 mM, while the K-M,K-app for N-lauroylglycine and longer chain N-acylglycines is in the range of 60 -90 mu M. For the amidation of N-acetylglycine, N-(tert-butoxycarbonyl)glyc ine, N-hexanoylglycine, and N-oleoylglycine, the rate of O-2 consumption is faster than the rate of glyoxylate production. These results indicate that there must be the initial formation of an oxidized intermediate from the N -acylglycine before glyoxylate is produced. The intermediate is shown to be N-acyl-alpha-hydroxyglycine by two-dimensional H-1-C-13 heteronuclear mult iple quantum coherence (HMQC) NMR.