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 (k(cat) values about 10(2) s(-1) and 10(3) 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 k(cat) values (12.1-1063 s(-1)), whereas for CinnAE, there were large differences in values for both Km (0.014-1.32 mM) and k(cat) (41.3-1410 s(-1)). Lability of the
ester bonds, as estimated from second-order
rate constants (k2) for chemical reaction with
sodium hydroxide, did not
correlate to k(cat) 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.
