Title: Thioesterases I and II of Escherichia coli. Hydrolysis of native acyl-acyl carrier protein thioesters.
Abstract: Thioesterases I and II of Escherichia coli hydrolyze the thioester linkages between fatty acid and CoA or acyl carrier protein (ACP). Previous workers had shown that chemically synthesized acyl-ACP substrates were hydrolyzed about 20-fold more slowly than the analogous CoA thioesters, and had suggested that the slower rate might be the result of modifications of the protein moiety which had occurred during the chemical synthesis. We have prepared a series of acyl-ACP substrates using acyl-ACP synthetase, a recently discovered enzyme which ligates long chain fatty acids to ACP. We have found that these preparations of native acyl-ACP are hydrolyzed much more slowly by both thioesterases than the analogous acyl-CoA or chemically synthesized acyl-ACP thioesters. The Michaelis constants (Km) of both thioesterases for native palmitoyl-ACP are 100 to 200 PM, a value over lo-fold higher than those reported for palmitoyl-CoA. The V,,, values of both enzymes for native palmitoyl-ACP are much lower than the values measured for palmitoyl-CoA. In the chemical procedure for acylating ACP, the amino groups are acetylated. This modification appears responsible for the differing rates of hydrolysis of native acyl-ACP and the substrates made chemically. Acetylation of our native palmitoyl-ACP increases the rate of hydrolysis of this substrate by both thioesterases. This modification of ACP is known to destabilize the tertiary structure of ACP under ionic conditions similar to those utilized in our assays. The greater rate of thioesterase cleavage of the chemically synthesized acyl-ACP over the native acyl-ACP can therefore be attributed to denaturation of the protein moiety of the former thioester.