Acylation is rate-limiting in glycosylasparaginase-catalyzed hydrolysis of N⁴-(4′-substituted phenyl)-l-asparagines

Abstract

Glycosylasparaginase catalyzes the hydrolysis of the N-glycosylic bond between N-acetyl-D-glucosamine and L-asparagine in the catabolism of glycoproteins. The mechanism has been proposed to resemble that of serine proteases involving an acylation step where a nucleophilic attack by a catalytic Thr residue on the carbonyl carbon of the N-glycosylic bond gives rise to a covalent beta-aspartyl-enzyme intermediate, and a deacylation step to give the final products. The question posed in this study was: Is the acylation step the rate-limiting step in the hydrolysis reaction as in serine proteases? To answer this question a series of mostly new substituted anilides was synthesized and characterized, and their hydrolysis reactions catalyzed by glycosylasparaginase from human amniotic fluid were studied. Five N4-(4'-substituted phenyl)-L-asparagine compounds were synthesized and characterized: 4'-hydrogen, 4'-ethyl, 4'-bromo, 4'-nitro, and 4'-methoxy. Each of these anilides was a substrate for the enzyme. Hammett plots of the kinetic parameters showed that acylation is the rate-limiting step in the reaction and that upon binding the electron distribution of the substrate is perturbed toward the transition state. This is the first direct evidence that acylation is the rate-limiting step in the enzyme-catalyzed reaction. A Brønsted plot indicates a small, negative charge (-0.25) on the nitrogen atom of the leaving group anilines containing electron-withdrawing groups, and a small, positive charge (0.43) on the nitrogen atom of the leaving group anilines containing electron-donating groups. The free energy (incremental) change of binding (delta deltaGb) in the enzyme-substrate transition state complexes shows that substitution of a substituted phenyl group for the pyranosyl group in the natural substrate results in an overall loss of binding energy equivalent to a weak hydrogen bond, the magnitude of which is dependent on the substituent group. The data are consistent with a mechanism for glycosylasparaginase involving rapid formation of a tetrahedral structure upon substrate binding, and a rate-limiting breakdown of the tetrahedral structure to a covalent beta-aspartyl-enzyme intermediate that is dependent on the electronic properties of the substituent group and on the degree of protonation of the leaving group in the transition state by a general acid.

Do N‐terminal nucleophile hydrolases indeed have a single amino acid catalytic center?

“…The Hammett plot can be divided into three linear segments: the points corresponding to substrates with electron‐donating substituents (R = CH 3 or OCH 3 ) lie on a line with a negative slope ρ = − 2.95 ± 0.98; for substrates with moderate electron‐withdrawing substituents (R = Br, CF 3 , COOC 2 H 5 or COCH 3 ) the slope is positive ρ = 0.90 ± 0.10; surprisingly, substrates with R = CN or NO 2 lie on a line with a negative slope ρ = −0.50 ± 0.18. The dependence of the rate on the electronic factor of the substituent for the PGA‐catalyzed hydrolytic reaction is very distinct as compared with the other Ntn hydrolases for which data are available [16,17]. The Hammett plot of the transpeptidation reaction catalyzed by γ‐glutamyl transpeptidase (GGT; EC 2.3.2.2), is biphasic, displaying a negative slope for the electron‐donating substituents ( ρ = −1.3) and a positive slope for the electron‐withdrawing substituents ( ρ = 0.4).…”

“…The glycosylasparaginase‐catalyzed hydrolysis [aspartylglucosaminidase (AGA), EC 3.5.1.26] is also characterized by a biphasic dependence: substrates with electron‐donating groups give a line with slope ρ = −0.94, and substrates with electron‐withdrawing groups give a line with slope ρ = 0.70. For all three enzymatic reactions (GGT‐catalyzed transpeptidation, and AGA‐catalyzed and PGA‐catalyzed hydrolysis), acylation is the rate‐limiting step [16–18]. However, the values of ρ for PGA are higher than those reported for glycosylasparaginase and GGT, indicating that the PGA‐catalyzed hydrolysis is much more sensitive to the electronic properties of the substituent on the leaving group.…”

Hydrolysis of phenylacetanilides catalyzed by penicillin G acylase from Alcaligenes faecalis: Sensitivity of the reaction to substitution in the leaving group

“…Reactions were performed in 0.1 M sodium phosphate buffer (pH 7.0), containing 10% DMSO (v/v); at T = 25°C and [S] 0 ) [E], and were followed spectrophotometrically. The Hammett plots of GGT, AGA and Ec-PGA feature a negative slope for substrates with electron-donating substituents and a positive slope for electron-withdrawing Rs [20,21]. A hypothesis was proposed that the breakdown of TI proceeds via concerted mechanism with the assistance of the protonated a-amino group of the N-terminal nucleophile, acting as the general acid.…”

“…The cleavage of the amidic C-N bond and the protonation of the leaving group N-atom occur simultaneously, but the substituent determines the geometry of this transition state (TS2, Scheme 2). Stronger electron-donating Rs favour higher degree of protonation of the leaving group in TS2, and electron acceptors favour higher degree of C-N bond cleavage [20,21]. At the active site of Ec-PGA a hydrogen bond between the leaving group nitrogen and the main-chain carbonyl of GlnB23 was found to favour the cleavage of the C-N bond, additionally stabilizing the partial negative charge on the leaving group nitrogen, rendering the protonation of the leaving group rate-limiting and displayed in the third linear segment of the Hammett plot of Ec-PGA [16].…”

“…Such kinetic investigations have been carried out with three other representatives of the Ntn-superfamily -c-glutamyl transpeptidase (GGT, EC 2.3.2.2), aspartylglucosaminidase (AGA, EC 3.5.1.26) and Ec-PGA [20,21,16]. For all three enzymatic [18].…”