The elucidation of the reaction mechanism of enzyme catalysis is an essential prerequi-site for the development of potent inhibitors. Structures for potential enzyme inhibitors could be derived from intermediates during enzyme catalysis or already optimized in-hibitor structures. The hQC is, due to their physiological and possibly pathophysiological function, a potential target for enzyme drug development. Therefore, the understanding of the catalytic mechanism of this enzyme is of great importance.
Two possible mechanisms of hQC catalysis were predicted in the past, which fundamen-tally differed in their modes of action [53]. First, the catalysis of the formation of a covalent intermediate (acyl-enzyme intermediate) could occur. In this mechanism a nu-cleophilic residue of the active site, for example an activated cysteine residue, attacks the γ-amide group of the N-terminal glutamine of the substrate. The acyl-enzyme intermedi-ate is formed and ammonia is released (acylation). In a second step, theα-amino group of glutamine attacks nucleophilically theγ-carbonyl. The product is released and the initial state is resumed (deacylation). Catalysis of hQC would be in this case in accordance with the mechanism of catalysis of serine/cysteine proteases or γ-glutamyl cyclotransferases.
The second alternative mechanism is based on a non-covalent reaction catalyzed by hQC (Fig. 4.6). Due to an intramolecular nucleophilic attack of the α-amino group at the γ-carbonyl of an N-terminal glutamine the substrate forms a tetrahedral, non-covalently enzyme-bound intermediate, which decomposes into ammonia and the pyroglutamyl pep-tide. Accordingly, hQC would exercise the catalytic function by binding of the substrate, which promotes a nucleophilic attack, and the stabilization of the tetrahedral intermediate.
Initial studies on hQC from pig pituitary suggested that the enzyme might possess thiol groups which are essential for catalysis [7]. However, identification of a disulfide bridge in hQC lead to exclusion of covalent catalysis with participation of cysteine residues [21].
In addition, glutaminyl cyclases of mammals showed no inhibition by serine modifying inhibitors, which supports the idea of a non-covalent catalysis [53].
O
Figure 4.6: Catalytic cycle of hQC catalyzing N-terminal glutaminyl substrates. First step is the formation of the Michaelis-Menten complex via binding of the substrate. Thereby it displaces the coordinated water molecule and occupies the fourth coordination site of the catalytic zinc. The catalytic zinc ion acts as a Lewis acid, pulls out electrons from theγ-carbonyl moiety of the N-terminal glutamine, thereby activating the γ-carbonyl carbon electrophilically. In addition, Glu201 activates via acid-base catalysis theα-amino group, which in turn gets more nucleophilic. Afterwards, theα-amino group performs a nucleophilic attack on theγ-carbonyl carbon, leading to a short-lived tetrahedral intermediate. Next, an intrinsic proton transfer to the potential leaving group via a conserved hydrogen bond network is performed to subsequently release ammonia and the product.
The enzyme catalyzes the stereospecifical cyclization of N-terminal glutaminyl residues in its physiological reaction and exhibits high selectivity for substrates with aromatic amino acids in the second N-terminal amino acid position [54]. Moreover, hQC showed a pH-dependence for N-terminal glutaminyl substrates, which are preferentially converted at
alkaline pH-values, especially at pH 8.0 [15]. It was shown in steady-state pH-dependency experiments that the Michaelis-Menten constant (KM) was influenced at pH-values above 8.0, indicating that hQC can bind the substrate only in the N-terminal deprotonated state.
The pH-dependence of the catalytic activity depends therefore on the protonation state of theα-amino group of the substrate.
Studies of the substrate specificity of hQC and its structural relationship to the zinc-dependent aminopeptidase family led to the first hypothesis of the mechanism of catalysis and the role of the metal ion [54]. The related zinc-dependent aminopeptidase has two metal ions within their active site, which resumes different tasks during catalysis. For in-stance, to polarize the scissile peptide bond by interacting with the carbonyl oxygen or to increase the nucleophilicity of the attacking water molecule. Most importantly, binding of the oxanion to stabilize the tetrahedral intermediate, which resulted from the nucleophilic attack. In the case of aminopeptidase from APAP, which has the highest structural homol-ogy to the animal glutaminyl cyclases, these tasks are performed mainly by one zinc ion in the active site, while the other metal ion is only used to fix the N-terminal amino nitrogen during catalysis. However, pyroglutamyl formation catalyzed by hQC differs compared to the mechanism of APAP. First, in contrast to the hydrolysis reaction of APAP, which removes the first N-terminal amino acid residue, the cyclization reaction of hQC is an intramolecular reaction. Second, the activation of the attacking nucleophile, which is an essential part in the catalytic cycle of APAP, is not necessary for hQC, because the free N-terminal nitrogen has a strong nucleophilicity. Despite these differences, partial reaction steps of the catalytic mechanisms of APAP and hQC could be the same. For example, in both cases the reaction is an addition elimination mechanism (SN2t). A zinc ion in the active site of hQC could therefore polarize theγ-carbonyl group of the substrate to increase the electrophilicity of the carbonyl carbon and, additionally, stabilize the oxanion interaction of the resulting tetrahedral intermediate after the nucleophilic attack of the α-amino group.
Crystal structures of APAP clearly showed two coordinated zinc ions in the active site, whereas for hQC only one zinc ion was identified even though the coordinating amino acid residues are highly conserved in both enzymes [16, 20]. As already mentioned, it
was assumed that the non-coordinating amino acids in hQC adopted new functions dur-ing evolution. This was demonstrated by site-directed mutagenesis experiments, in which substitution resulted in a non-functional enzyme (Asp248) or reduced catalytic activity (His140) [55]. Due to the position of Asp248 in the crystal structure it was concluded that it forms together with Glu201 and Asp305 a conserved hydrogen bond network, which is crucial for proton shuttling from theα-amine group of the tetrahedral intermediate to the γ-amide amino group (Fig. 4.6).
Nevertheless, a detailed reconstruction of the catalytic steps of the reaction is not yet possible. For instance, the short-lived tetrahedral intermediate is only postulated, but so far not experimentally confirmed.