Characterization of discrete mechanistic steps in the trajectory of QCs

The chemical reaction from glutamine to pyroglutamic acid is assigned to the addition-elimination mechanism SN2t [60]. This bimolecular reaction is characterized by the concerted addition of a nucleophile to a polarized molecule and the release of a leaving group (elimination). During the reaction a short-lived tetrahedral transition state occurs. Considering the cyclization reaction of glutamine, the nucleophile and the polarized group belong to the same molecule. The QC enhances this intra-molecular ring closure reaction by the activation of both reacting moieties. In the following section the discrete steps of the QC catalysis are discussed.

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4.2.2.1 Chemical activation of substrate moieties support the nucleophilic attack

Figure 4.3 Postulated reaction cycle of Zn(II)-dependent QC The postulated catalytic cycle of glutaminyl cyclase depicts the formation of a tetrahedral reaction intermediate after the α-amino group has attacked the γ-carbonyl nucleophilically. This intermediate breaks down into ammonia and pyroglutamic acid.

Once the substrate is bound to the enzyme, it is suggested that it becomes activated from two sides (cf. Figure 4.3). As shown in Section 3.6.2 and Figure 3.17, the EPR data obtained with the Co(II)-isoDromeQC wild type after several incubation periods depict a strong shift of the signal maximum after 10 ms. This shift is at its maximum after 160 ms. As described above, the substrate binding can already be observed after 10 ms. Almost simultaneously with the detected deformation of the coordination geometry of Co(II) induced by substrate binding, a further increase of the deformation occurs. This indicates that electron(s) from Co(II) become strongly delocalized along the Z-axes of the Co(II) coordination sphere. According to the postulated mechanism, this electron delocalization would occur along the axis of the carbonyl carbon - γ-carbonyl oxygen and the Co(II) ion. This non-covalent metal-oxanion interaction effectuates a polarization of the γ-carbonyl carbon and increases its electrophilic force. Simultaneously, as indicated in the crystal structure (cf. Figure 3.11), it is most likely that the γ-carboxylic group of E190 abstracts a proton from the -amino group of the N-terminal glutamine. This deprotonation step enhances the already high nucleophilicity of the -amino nitrogen. [73, 90].

Presumably, the consequence is that the α-amino nitrogen attacks the γ-carbonyl and forms a

Zn²⁺

117 between the nucleophile and the polarized carbon atom is still incomplete. The bond between the leaving group and the carbon atom is prolonged but still existent. The tetrahedral intermediate is possibly stabilized by the catalytically active metal ion. Currently the existence of this intermediate state is likely but remains for now hypothetical.

The question of whether the formation or decomposition of the tetrahedral intermediate is rate-limiting was investigated by Dr. Franziska Seifert employing beta-secondary isotope effect experiments (results see Supplemental Section 8.8). For this trial the deuterated Q-AMC (cf.

Figure 4.4 A) was used as substrate for the fluorescence spectroscopic assay (cf. Section 2.6.2).

Characteristic for -secondary isotope effects is that the moiety harboring the isotope, in this case deuterated methylen groups, are in the proximity of these moieties which are involved in bond formation. This experiment can unveil differences in the activation energies that are required for the formation or stabilization of the tetrahedral intermediate when this step is rate limiting. The stabilization of this tetrahedral intermediate is most likely due to hyperconjugation.

In this case an electron from the orbital of the σ-bond between the -carbon atom and the hydrogen or deuterium stabilizes the polarized γ-carbon by mesomeric effect (cf. Figure 4.4. B).

Hyperconjugation and the related stabilizing effects to transition states are a source of -secondary isotope effects [98]. In case of a deuterated δ-methylen group the stabilization of the transition state requires higher activation energy. This in turn leads to lower reaction velocity.

The quotient of the determined kinetic constants of the reaction with deuterated and undeuterated substrate corresponds to the equilibrium isotopic effect (KM) and the kinetic isotopic effect (kcat). The isotopic effects become significant in a range between 1.5 and 7. The determined isotopic effects were below 1.5 and consequently not significant. Hence, we can conclude that the formation or the decomposition of the suggested tetrahedral intermediate is most likely not the rate-limiting step.

4 Discussion

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Figure 4.4 Deuterated fluorogenic substrate Q-AMC and the postulated tetrahedral intermediate (A) The fluorogenic substrate was utilized to examine a beta-secondary isotope effect employing fluorescence spectroscopy. (B) The postulated tetrahedral intermediate is possibly stabilized by hyperconjugation. In case of deuterated methylene groups, the hyperconjugation requires higher activation energy compared to hydrogen-containing groups. When the formation of this intermediate is rate-limiting, this effect causes lower rate constants.

4.2.2.2 The release of ammonia (deamination) is accompanied by the decomposition of the reaction intermediate

The EPR data of Co(II)-isoDromeQC wild type incubated with QQ after several incubation times show that the shift of the signal maximum after 2 s becomes smaller compared to the signal after 160 ms. This indicates that the strong distortion in the coordination sphere of Co(II) is reconstituted. This relaxation can be induced by the decomposition of the putative tetrahedral intermediate. This step includes the cleavage of the γ-amino moiety and the removal of the delocalized electron (cf. Figure 4.3). Crystal structures of Co(II)-isoDromeQC wild type (cf. Figure 3.11 B and C) depict that the γ-carboxylic moiety of D228 and γ-amino nitrogen are in hydrogen bond distance. It is suggested that this amino acid provides a proton to the leaving γ-amino group. This proton transfer possibly enhances the elimination of ammonia. The release of ammonia makes the cyclization reaction quasi-irreversible. Schilling et al. al showed that ammonia concentration up to 50 mM does not inhibit the reaction [80]. The missing product inhibition indicates that the deamination step is most likely not rate-limiting.

+H₃N