Methyl Phenylalkanoates as Substrates to Probe the Active Sites of Esterases

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Abstract
We have used methyl esters of phenylalkanoic acids to probe the active site of two esterases (FAE‐III and CinnAE) from Aspergillus niger. Only methyl 4‐hydroxy‐3‐methoxycinnamate and 4‐hydroxy‐3‐methoxyphenylpropionate out of 19 substrates tested were significant substrates for both enzymes (kcat values about 102 s−1 and 103 s−1, respectively). Lengthening or shortening the aliphatic side chain while maintaining the same aromatic substitutions completely abolished activity for both enzymes, which demonstrates the importance of the correct distance between the aromatic group and the ester bond. Differences in Km values for FAE‐III were small (0.45–2.08 mM) but there were two orders of magnitude difference in Kcat values (12.1–1063 s−1), whereas for CinnAE, there were large differences in values for both Km (0.014–1.32 mM) and kcat (41.3‐1410 s−1)‐ Lability of the ester bonds, as estimated from second‐order rate constants (A:2) for chemical reaction with sodium hydroxide, did not correlate to kcat for CinnAE (r= 0.33) or for FAE‐III (r= 0.43). Maintaining the phenylpropenoate structure but altering the substitutions on the aromatic ring demonstrated the following: a 3‐methoxy group is essential for FAE‐III activity, whereas a 3‐methoxy group precluded activity of CinnAE, with the exception of methyl 4‐hydroxy‐3‐methoxycinnamate which was a relatively poor substrate for CinnAE; (b) increasing the number of methoxy substitutions increased the activity of FAE‐III, and decreased the activity of CinnAE; (c) 4‐hydroxy substituents, and additional hydroxy substituents, increased the activity of CinnAE, but decreased that of FAE‐III; (d) the rate of hydrolysis with sodium hydroxide of the methyl esters in general is decreased by hydroxy substitutions on the aromatic ring but increased by methoxy substitutions. Analysis of kinetic data obtained in the presence of inhibitors indicated that substrate analogs were able to bind to both free CinnAE and to a CinnAE‐substrate complex, but conversely, were only able to bind to free FAE‐III. The results show that the specificities of the two A. niger esterases are complementary. The rate of hydrolysis by this class of carboxylic ester hydrolase does not depend on the intrinsic lability of the ester bond, but depends on both the distance between the aromatic ring and the ester bond, and the substitutions on the aromatic ring.