Glycosylasparaginase (GA) catalyzes the hydrolysis of the N-glycosylic bond in β-N-acetylglucosaminyl-L-asparagine to give N-acetylglucosamine, aspartic acid and ammonium ion in the catabolism of N-linked glycoproteins. A deficiency or absence of activity of GA gives rise to the most common inherited disorder of glycoprotein metabolism, aspartylglycosaminuria. The mechanism for GA has been proposed to be analogous to that of serine proteases, involving an acylation reaction to an acyl-enzyme intermediate, followed by a deacylation reaction. Studies of the mechanism thus far show that some properties of GA are similar to properties of serine proteases, although differences exist in some of the properties. Serine proteases have esterase activity where the rate-limiting step in the hydrolysis of substituted phenyl esters is the deacylation step. The esterase activity of GA was investigated using O4-(parasubstituted phenyl)-L-aspartate esters synthesized for the first time. The release of the parasubstituted phenolate was measured upon incubation with GA from human amniotic fluid. The rates of hydrolysis of the esters were dependent on the electronic properties of the parasubstitution. The rate-determining step in the hydrolysis of the esters by GA is the acylation step, opposite to that of serine proteases. The analogy of the mechanisms for serine proteases and GA is not absolute. The research was supported in part by funds provided by The University of North Carolina at Charlotte.

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.

Glycosylasparaginase catalyzes the hydrolysis of the N-glycosylic bond between asparagine and N-acetylglucosamine in the catabolism of N-linked glycoproteins. Previously only three competitive inhibitors, one noncompetitive inhibitor, and one irreversible inhibitor of glycosylasparaginase activity had been reported. Using human glycosylasparaginase from human amniotic fluid, L-aspartic acid and four of its analogues, where the alpha-amino group was substituted with a chloro, bromo, methyl or hydrogen, were competitive inhibitors having Ki values between 0.6-7.7 mM. These results provide supporting evidence for a proposed intramolecular autoproteolytic activation reaction. A proposed phosphono transition state mimic and a sulfo transition state mimic were competitive inhibitors with Ki values 0.9 mM and 1.4 mM, respectively. These results support a mechanism for the enzyme-catalyzed reaction involving formation of a tetrahedral high-energy intermediate. Three analogues of the natural substrate were noncompetitive inhibitors with Ki values between 0.56-0.75 mM, indicating the presence of a second binding site that may recognize (substituted)acetamido groups.

Glycosylasparaginase catalyzes the hydrolysis of the N-glycosylic bond in N4-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-l-asparagine in the catabolism of N-linked oligosaccharides. A deficiency, or absence, of enzyme activity gives rise to aspartylglycosaminuria, the most common disorder of glycoprotein metabolism. The enzyme catalyzes the hydrolysis of a variety of asparagine and aspartyl compounds containing a free α-carboxyl group and a free α-amino group; computational studies suggest that the α-amino group actively participates in the catalytic mechanism. In order to study the importance of the α-carboxyl group and the α-amino group on the natural substrate to the reaction catalyzed by the enzyme, 14 analogues of the natural substrate were studied where the structure of the aspartyl group of the substrate was changed. The incremental binding energy (ΔΔGb) for those analogues that were substrates was calculated. The results show that the α-amino group may be substituted with a group of comparable size, for the α-amino group contributes little, if any, to the transition state binding energy of the natural substrate. The α-amino group position acts as an “anchor” in the binding site for the substrate. On the other hand, the α-carboxyl group is necessary for enzyme activity; removal of the α-carboxyl group or changing it to an α-carboxamide group results in no hydrolysis reaction. Also, N-acetyl-d-glucosamine is not sufficient for binding to the active site for efficient hydrolysis by the enzyme. These results provide supporting evidence for a proposed intramolecular autoproteolytic activation reaction for the enzyme. However, the results raise a question as to an important role for the α-amino group in the catalytic mechanism as indicated in computational studies.

The syntheses of three analogues of N4-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-l-asparagine are described. N-(2-Acetamido-2-deoxy-β-d-glucopyranosyl)succinamide was synthesized by the reaction of pentafluorophenyl succinamate with 2-acetamido-2-deoxy-β-d-glucopyranosylamine. 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-d-glucopyranosylamine was synthesized, and the complete assignment of the 1H NMR spectrum is given. Reaction of the protected β-d-glycosylamine with l-malic acid chloralid in the presence of a coupling agent (EEDQ) gave N4-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-d-glucopyranosyl)-l-malamic acid chloralid that was deprotected two ways: (1) using ammonia, which gave N4-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-l-2-hydroxysuccinamide, and (2) using hydrazine, which gave N4-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-l-2-hydroxysuccinamic acid hydrazide.