METHYL PHENYLALKANOATES AS SUBSTRATES TO PROBE THE ACTIVE-SITES OF ESTERASES

We have used methyl esters of phenylalkanoic acids to probe the active site of two esterases (FAE-III and CinnAE) from Aspergillus niger. On ly methyl 4-hydroxy-3-methoxycinnamate and 4-hydroxy-3-methoxyphenylpr opionate out of 19 substrates tested were significant substrates for b oth enzymes (k(cat) values about 10(2) s(-1) and 10(3) s(-1), respecti vely). Lengthening or shortening the aliphatic side chain while mainta ining the same aromatic substitutions completely abolished activity fo r both enzymes, which demonstrates the importance of the correct dista nce between the aromatic group and the ester bond. Differences in K-m 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 f or CinnAE, there were large differences in values for both K-m (0.014- 1.32 mM) and k(cat) (41.3-1410 s(-1)). Lability of the eater bonds, as estimated from second-order rate constants (k(2)) for chemical reacti on with sodium hydroxide, did not correlate to k(cat) for CinnAE (r = 0.33) or for FAE-III (r = 0.43). Maintaining the phenylpropenoate stru cture but altering the substitutions on the aromatic ring demonstrated the following: a 3-methoxy group is essential for FAE-III activity, w hereas a 3-methoxy group precluded activity of CinnAE, with the except ion of methyl 4-hydroxy-3-methoxycinnamate which was a relatively poor substrate for CinnAE; (b) increasing the number of methoxy substituti ons increased the activity of FAE-III, and decreased the activity of C innAE; (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 a ble to bind to both free CinnAE and to a CinnAE-subatrate 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 d oes not depend on the intrinsic lability of the ester bond, but depend s on both the distance between the aromatic ring and the eater bond, a nd the substitutions on the aromatic ring.