Substrate-derived reversible and irreversible inhibitors of the multi-enzyme I of gramicidin S biosynthesis

Abstract

1. 1. The heavy fraction (Fraction I enzyme) which activates proline, valine, ornithine and leucine in gramicidin S biosynthesis has been purified by preparative liquid isoelectric focusing. 2. 2. l-Isoleucine, an amino acid not found in gramicidin S, supported PP i-ATP exchange to 43% that of l-leucine. 3. 3. Of the ring analogues of proline tested, l-azatidine-2-carboxylic acid and l-pipecolic acid supported PP i-ATP exchange to the extent of 64 and 13% that of proline, respectively. 4. 4. In the ring substituted analogues of proline, trans-3-methylproline and trans-4-fluoroproline inhibited exchange in the presence of l-proline by 29 and 93%, respectively. 5. 5. The d-isomers of the constituent amino acids of gramicidin S did not support PP i-ATP exchange and at equimolar concentrations inhibited the exchange supported by the l-isomers by 80%. 6. 6. An analysis of the slope-intercept effects on double reciprocal plots obtained when ATP is the variable substrate and l-proline is the changing fixed substrate gives results which are in accord with a random order of addition for ATP and l-proline to the heavy fraction. 7. 7. The chloromethyl ketone of l-proline (Pro-Cmk) inhibited the PP i-ATP exchange occuring in the presence of l-proline of Fraction I enzyme 95% in 60 min. Preincubation for 60 min with Pro-Cmk resulted in a residual activity of 9.7% whereas the native enzyme alone and in the presence of the reversible inhibitor l-2-hydroxymethyl pyrrolidine gave a residual activity of 73 and 79%, respectively. The exchange activity supported by l-valine, l-ornithine and l-leucine remained essetially unchanged.