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Enzymatic Hydrolysis of Tertiary Amide Bonds by anti Nucleophilic Attack and Protonation Per-Olof Syreń * School of Engineering Sciences in Chemistry, Biotechnology and Health, Science for Life Laboratory, Department of Fibre and Polymer Technology, and Department of Protein Science, KTH Royal Institute of Technology, Solna, Sweden 17165
J. Org. Chem. 2018.83:13543-13548. Downloaded from pubs.acs.org by 46.243.173.123 on 11/02/18. For personal use only.
S Supporting Information *
ABSTRACT: The molecular mechanisms conferring high resistance of planar tertiary amide bonds to hydrolysis by most enzymes have remained elusive. To provide a chemical explanation to this unresolved puzzle, UB3LYP calculations were performed on an active site model of Xaa-Pro peptidases. The calculated reaction mechanism demonstrates that biocatalysts capable of tertiary amide bond hydrolysis capitalize on anti nucleophilic attack and protonation of the amide nitrogen, in contrast to the traditional syn displayed by amidases and proteases acting on secondary amide bonds.
A
mides are ubiquitous structural and functional units in synthetic chemistry, medicine, and biology.1,2 Substituents on the nitrogen atom impact reactivity,1,3,4 cis/trans isomerization ratios,5 and the capability of amide bonds to participate in hydrogen bonding interactions that are instrumental in enabling helices and other basal structural motifs. The purpose of this work was to seek a chemical explanation to why tertiary amide bonds pose unsurmountable challenges for most amidases and proteases, a fact that gives amide bonds involving the nitrogen atom of proline a prominent position in cell biology in regulating stability and half-life of proteins and peptides.6−8 Herein, it is shown that biocatalysts capable of efficient hydrolysis of planar, proline-based tertiary amide bonds uniquely position the nucleophile and the general acid on opposite faces of the reacting amide group. When incorporated into proteins, 19 of the 20 standard proteinogenic amino acids form secondary amide bonds that reside almost9 exclusively in trans configuration. In contrast, for the 20th amino acid proline, the nitrogen and Cα atoms are incorporated into a pyrrolidine ring, which give amide bonds that lack a hydrogen atom. This arrangement allows prolinecontaining tertiary amide bonds to adapt both cis and trans configuration in proteins7 and enables the formation of collagen and other unique structural features.10,11 In addition to affecting macromolecular structure, the chemical reactivity of tertiary amide bonds is generally reduced12,13 compared to that of secondary amides, which is partly reflected in relative nucleophilicity of corresponding amines.4 Tertiary amide bonds, involving the nitrogen atom in the pyrrolidine ring of proline and other N-heterocycles, prevail in drugs14−17 (Figure 1). Furthermore, these bonds occur in twisted amides that are of considerable interest in synthetic organic chemistry.18 Tertiary amide bonds are abundant in Nature and can, for instance, in addition to proline-containing amides, be formed by N-methylation of both © 2018 American Chemical Society
Figure 1. Pyrrolidine and piperidine ring systems are highlighted in blue. For reference, backbone N-methylation in the natural product griselimycin (1d) is shown in red.14−17
nonribosomal peptides and ribosomally synthesized peptides,19,20 a process key to tune dynamics and bioactivity of natural products.21 Design22 and generation of artificial oligomers and polymers based on tertiary amide bonds, in particular, oligo-Nsubstituted glycines (also referred to as peptoids23,24), is receiving significant attention in organic chemistry, material science, biotechnology, and medicine.25−28 Received: August 8, 2018 Published: September 26, 2018 13543
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The Journal of Organic Chemistry
lone pair will point away from the catalytic base, which in current models resides on the same face38 of the amide bond as the enzyme nucleophile (Figure 2a,b). This organization allows activation of the nucleophile at the onset of the reaction sequence but precludes progression of catalysis from the first tetrahedral intermediate by proton transfer to the reacting nitrogen (for which the catalytic base formally acts as general acid). For this reason, nitrogen inversion is key to generate a catalytically competent second tetrahedral intermediate; one that displays the lone pair in a flipped and productive orientation for proton abstraction. Previous studies have shown how amidases and proteases universally use the available hydrogen on the reacting nitrogen atom of secondary amide bonds, to stabilize the highenergy transition state for nitrogen inversion by H-bonding (Figure 2b).39,40 As tertiary amide bonds lack a hydrogen atom, it is hypothesized herein that they must undergo enzymatic hydrolysis by other reaction mechanisms. Previous work has shown that for β-lactam substrates, with the distorted pyramidal nitrogen (0.4 Å above the plane for penicillins41) already pointing toward the syn enzyme nucleophile and Ser catalytic base in the ground state,42 a proton shuttle operates to deliver a proton to the lone pair without inversion (Figure 2c). The scarce family of Xaa-Pro peptidases capable of hydrolyzing trans proline-based tertiary amide bonds35,43 includes aminopeptidase P acting on polyamides (also referred to as X-prolyl aminopeptidase) and prolidases acting on dipeptide substrates.35,43,44 Although Xaa-Pro peptidases can display different domain architectures, they share a dinuclear active site embedded in a catalytic domain composed of a curved β-sheet referred to as the “pita bread” fold.44−51 The bimetallic catalytic center, typically composed of two manganese atoms in oxidation state +2, displays overall conserved44 amino acid ligands within this enzyme superfamily. The metals interact with a nucleophilic μ-hydroxide activated by a catalytic glutamate, both of which reside on the same face of the peptide bond.35,43,44 Interestingly, initial visual inspection of available structures of Escherichia coli aminopeptidase P complexed with inhibitors (PDB 1A1649 and 1N5152) revealed a histidine (H243) in spatial arrangement that would fulfill the geometrical criterion for the general acid shown in Figure 2d. The fact that the H243A mutant of E. coli aminopeptidase P is catalytically inactive but still displays productive substrate binding53 supports a previously unknown catalytic role of this key residue that is absolutely conserved in all currently known Xaa-Pro peptidases.44,50,51,54 In fact, mutation of this residue in human prolidase is predicted to be highly deleterious according to the consensus classifier PredictSNP55 (87% confidence score). Notably the location of the histidine on the distal side of the scissile amide bond in relation to the hydroxide nucleophile is in sharp contrast to all other families of proteases.38 According to this unique concept, nitrogen inversion, which is added on top of the high-energy tetrahedral intermediate,9 can be avoided. To explore this catalytic path, a “theozyme”56 model of the active site (also referred to as cluster model57) comprising the bimetallic Mn2+58 catalytic center, the glutamate interacting with the hydroxide nucleophile, the proposed general acid histidine, and an Ala-Pro model substrate was generated based on the high-resolution crystal structure of empty human cytosolic X-prolyl aminopeptidase (1.6 Å, PDB 3CTZ44). It is known that the histidine suggested herein to act as a general acid changes orientation by swinging inward toward the bimetallic center upon substrate binding in prolidases51 and X-prolyl aminopeptidases.48,52 For that reason, the coordinates of the proposed key histidine were taken from the E. coli enzyme
Proteolytic stability mediated by tertiary amide bonds is of considerable interest from a drug development perspective.29 It is well-known that alkylating backbone nitrogen atoms to form tertiary amide bonds in peptoids,23,24,30 peptidomimetics,31,32 and peptides6,7,21,33,34 significantly slows down, or even completely abolishes, enzymatic hydrolysis. A recent study demonstrated that methylation of the targeted P1′ amide nitrogen in model substrates enforced hydrolysis by proteases at a distal site.34 This catalytic trend is perhaps further well illustrated by the fact that the proline-containing tertiary amide bond in the antihypertensive drug Captopril (Figure 1c) remains to a large extent metabolically intact after administration.6 In fact, several metabolized peptides display a proline in the position penultimate to the N-terminus.35 The molecular reasons underpinning the high resistance of proline-based and other tertiary amide bonds toward most hydrolytic enzymes have remained elusive. One hypothesis is that alteration of the conformational landscape and dynamics of the peptide substrate upon nitrogen alkylation would interfere with enzyme catalysis. However, whereas N-alkylation generally renders peptoids more flexible than their parent peptides, the situation can be opposite for other peptidomimetics33 as well as for proline-rich regions that function as backbone rigidifiers on the local protein structural level.36 Stereoelectronic effects are of high relevance for amide bond hydrolysis: n to σ* interactions enforce the lone pair on the reacting nitrogen atom to be situated antiperiplanar37 to the bond formed between the incoming nucleophile and the former carbonyl carbon in the first tetrahedral intermediate generated during hydrolysis (Figure 2a). In this spatial arrangement, the
Figure 2. Reaction mechanisms. (a) The syn spatial location of the nucleophile (Nu) and catalytic base (BH) relative to the reacting amide bond is schematically depicted for the first tetrahedral intermediate formed during hydrolysis of secondary amide bonds by amidases/ proteases. The orientation of the lone pair of the reacting nitrogen atom is shown. (b) Key hydrogen bond donated by the reacting NH group is shown by the arrow for a serine protease catalytic machinery. The nitrogen lone pair that flips orientation is faded. (c) Two catalytic serines of class A β-lactamases enable syn nucleophilic attack and proton shuttling to the pyramidal reacting nitrogen atom. The nitrogen lone pair of the distorted amide bond in the ground state is depicted. (d) The anti nucleophilic attack and protonation for planar tertiary amide bonds. The scissile amide bond is shown in magenta. 13544
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Figure 3. Energy profile diagram for tertiary amide bond hydrolysis (see Supporting Information for details). Transition-state structures are schematically shown with bonds formed/broken in dashed. Hydrogen bonds are represented by dotted lines. For clarity, some catalytic amino acids are labeled in the first TS structure (numbering corresponds to E. coli aminopeptidase P), with the for amidases uniquely positioned general acid labeled in bold. Relative energies are given at the UB3LYP/6-311G++(2d,2p)+LANL2DZ level of theory with PCM (ε = 2). Values given include dispersion and thermal correction to the internal energy (at 298 K).
catalytic glutamate allow for generation of a proline with a charged nitrogen leaving group, aligned with the relatively high pKa of proline. Furthermore, the mechanistic calculations stressed the importance of H361 in stabilizing the oxyanion by proton transfer. The calculated activation barrier of 16.9 kcal/mol is close to the experimentally determined value of 15 kcal/mol for a tripeptide substrate (kcat of 85 s−1).47 Transfer of the hydroxide proton to the catalytic E383 likely occurs prior to protonation of the nitrogen lone pair, as protonation of the corresponding gemdiol intermediate (path B, Figure 3) was associated with a higher energy. Nitrogen inversion (path C, Figure 3) resulted in a prohibitively high barrier of activation and resided 10 kcal/mol on top of the corresponding tetrahedral intermediate. This finding is aligned with the known fact that nitrogen inversion in ring systems is energetically demanding.64 Previous work on human methionine aminopeptidase, a biocatalyst adopting the pita-bread fold and acting on polypeptides with methionine in the N-terminus, suggested that the histidine acting as oxyanion hole (corresponding to H361 in Figure 3) could serve as proton donor to the reacting nitrogen atom.65 The QM calculations herein show a large distance of 4.7 Å between the two relevant nitrogen atoms, which renders associated proton transfer unlikely. Another study emphasized a potential role of the herein suggested key histidine (corresponding to H243 in Figure 3) to orient the substrate for catalysis, by binding in its neutral form to the reacting nitrogen atom.46 The histidine has been discussed to interact with the substrate P1′ carbonyl oxygen in aminopeptidase P66 or the terminal carboxylate for prolidases.51 To further support the unprecedented catalytic role of the histidine suggested herein, attention was given to promiscuous prolidase activity displayed by some organophosphate hydrolases.67 The organophosphate acid
(i.e., H243 in aminopeptidase P) complexed with substrate analogue. Superposition of the human and bacterial enzymes over identical metal ligands (rmsd of 0.4960 Å when superposing the structures 3CTZ44 and 1N5152) allowed for dissection of the histidine geometry in a productive conformation for catalysis, while retaining the dinuclear center of higher resolution from the human structure. Geometry optimization of the ES complex in the final theozyme consisting of 134 atoms (Figure S1) was performed by quantum mechanical (QM) calculations, using density functional theory and evaluating two commonly used hybrid functionals: B3LYP59−62 and M06-2X.63 As expected, due to five unpaired d-electrons situated on each manganese, QM calculations on a closed-shell system resulted in anomalous metal−metal distance (2.72 Å, Table S1). Therefore, high-spin unrestricted DFT calculations were used using the 6-31G(d,p)+LANL2DZ mixed basis set (see Supporting Information). As the two metal atoms display weak antiferromagnetic coupling (J close to −2 cm−1),58 this strategy was considered an adequate approximation. UB3LYP resulted in a Mn−Mn distance of 3.30 Å, which was in agreement with the average 3.34 ± 0.07 Å obtained from crystallographic data, whereas UM06-2X yielded a significantly shorter intermetal distance of 3.22 Å. Thus, UB3LYP was used for subsequent calculations, which did not give rise to significant spin contamination according to evaluation of the total spin-squared operator (S2 of 30.007, expected value is 30.000). The calculated reaction mechanism (Figure 3; for a full reaction scheme, see Figure S2) supported the hypothesized catalytic role of the histidine in delivering a proton from the opposite face of the amide bond in relation to the nucleophile. The transition state for proton transfer (TS3, Figure 3), with the reacting nitrogen on top of the envelope, was found to be overall rate-limiting. Proton transfers by histidine and the 13545
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anhydrolase from Alteromonas sp. displays a histidine in a spatial arrangement to act as general acid according to the model in Figure 2d. Whereas this biocatalyst shows high promiscuous prolidase activity, the Pseudomonas diminuta phosphotriesterase (PTE), for which the key histidine is replaced by a Ser (Figure S3), is inert toward tertiary amides.67 Alignment of empty (PDB 1I0B68) and substrate-bound PTE (PDB 1EZ269) demonstrates that this Ser does not undergo backbone conformational change upon substrate binding. This fact indicates that the mechanism herein involving an anti His general acid that delivers a proton from solvent to the reacting nitrogen atom likely requires multiple amino acid substitutions during evolution to enable the required dynamics. In chemistry, hydrolysis of 2-carboxy-N,Ndiethylnaphthalamic acid proceeds 35-fold faster compared to that of the corresponding monocarboxylate.70 The fact that this finding can be ascribed to a general acid residing on the opposite face of the amide bond than the intramolecular nucleophile70 stresses the importance of the anti mechanism (Figure 2d) in both fundamental organic chemistry and biology. Enhancing our current limited understanding of fundamental proline biochemistry is of high relevance for metabolic pathways of microbial communities,71 for the recently discovered Pro/Nend rule pathway that targets proteins for destruction2 and in tumor progression and disease.72 Proline-rich regions have a high abundance in intrinsically disordered proteins73 and oligoand polyprolines display interesting material properties as antifreeze agents74 and as cell-penetrating materials.75 Moreover, proline and derivatives thereof constitute building blocks for novel polymers.76,77 Proline has been described as a privileged scaffold in medicinal chemistry17 and in the de novo design and generation of peptide macrocycles.28 Prolidase deficiency, an autosomal recessive disease with severe effects, is associated with lack of capability in degrading Xaa-Pro dipeptides.35,43,78 As Xaa-Pro peptidases can display significant promiscuous activity toward N-methylated amino acids in the P1′ position,47 anti nucleophilic attack and protonation could play an important role in enabling hydrolysis of tertiary amide bonds.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.joc.8b02053. Computational details, reaction mechanism in full and coordinates of all intermediates and transition states (PDF) Data file containing transition-state structures in .xyz format (ZIP)
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Note
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Per-Olof Syrén: 0000-0002-4066-2776 Notes
The author declares no competing financial interest.
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ACKNOWLEDGMENTS The author greatly acknowledges financial support of this work by a FORMAS young research leader fellowship (Grant No. 2017-01116). The PDC Center for High Performance Computing at the Royal Institute of Technology (KTH) is greatly acknowledged. 13546
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