Mechanisms of enzymatic and acid-catalyzed decarboxylations of prephenate

Abstract

The prephenate dehydrogenase activity of the bifunctional enzyme chorismate mutase-prephenate dehydrogenase from Escherichia coli catalyzes the oxidative decarboxylation of both prephenate and deoxoprephenate, which lacks the keto group in the side chain (V 78% and V/K 18% those of prephenate). Hydride transfer is to the B side of NAD, and the acetylpyridine and pyridinecarboxaldehyde analogues of NAD have V/K values 40 and 9% and V values 107 and 13% those of NAD. Since the 13C isotope effect on the decarboxylation is 1.0103 with deuterated and 1.0033 with unlabeled deoxoprephenate (the deuterium isotope effect on V/K is 2.34), the mechanism is concerted, and if CO2 has no reverse commitment, the intrinsic 13C and deuterium isotope effects are 1.0155 (corresponding to a very early transition state for C-C bond cleavage) and 7.3, and the forward commitment is 3.7. With deoxodihydroprephenate (lacking one double bond in the ring), oxidation occurs without decarboxylation, and one enantiomer has a V/K value 23-fold higher than the other (deuterium isotope effects are 3.6 and 4.1 for fast and slow isomers; V for the fast isomer is 5% and V/K 0.7% those of prephenate). The fully saturated analogue of deoxoprephenate is a very slow substrate (V 0.07% and V/K approximately 10(-5%) those of prephenate). pH profiles show a group with pK = 8.3 that must be protonated for substrate binding and a catalytic group with pK = 6.5 that is a cationic acid (likely histidine). This group facilitates hydride transfer by beginning to accept the proton from the 4-hydroxyl group of prephenate prior to the beginning of C-C cleavage (or fully accepting it in the oxidation of the analogues with only one double bond or none in the ring). In contrast with the enzymatic reaction, the acid-catalyzed decarboxylation of prephenate and deoxoprephenate (t1/2 of 3.7 min at low pH) is a stepwise reaction with a carbonium ion intermediate, since 18O is incorporated into substrate and its epi isomer during reaction in H218O. pH profiles show that the hydroxyl group must be protonated and the carboxyl (pK approximately 4.2) ionized for carbonium ion formation. The carbonium ion formed from prephenate decarboxylates 1.75 times faster than it reacts with water (giving 1.8 times as much prephenate as epi isomer). The observed 13C isotope effect of 1.0082 thus corresponds to an intrinsic isotope effect of 1.023, indicating an early transition state for the decarboxylation step. epi-Prephenate is at least 20 times more stable to acid than prephenate because it exists largely as an internal hemiketal.(ABSTRACT TRUNCATED AT 400 WORDS)

The Use of Isotope Effects to Determine Enzyme Mechanisms

“…6-Phosphogluconate dehydrogenase was also shown to catalyze a stepwise reaction (28), but prephenate dehydrogenase provided some surprises (29). The isotope effects were measured with a substrate lacking the keto group in the side chain but having a V max of 78% and V/K of 18% that of prephenate.…”

Crystal Structure of Prephenate Dehydrogenase from Aquifex aeolicus

“…The C-4 of prephenate is considered the reactive center, since the hydride at this position is transferred to NAD ϩ during the enzymatic reaction (42,43).…”

Metabolic engineering of a genetic selection system with tunable stringency

“…In contrast, under selective conditions, aromatic residues become enriched at position 14 as the selection stringency is increased upon raising the tetracycline concentration. conceivably stabilize the cationic intermediate (21) that is generated upon decarboxylation of prephenate through cationinteractions (22), but a structural role may be more plausible, given the observed bias for phenylalanine over tryptophan and tyrosine, which are normally preferred at cation-binding sites, and the appearance of histidine, which is never used for this purpose (23). Structures of the enzyme complexed with substrate or transition state analogs will be needed to clarify the precise nature of this interaction.…”