Báo cáo khoa học: Kinetic studies and molecular modelling attribute a crucial role in the specificity and stereoselectivity of penicillin acylase to the pair ArgA145-ArgB263

Kinetic Studies and Molecular Modelling Attribute a Crucial Role in the Specificity and Stereoselectivity of Penicillin Acylase to the Pair ArgA145-ArgB263

Tác giả: Maya Guncheva, Ivaylo Ivanov, Boris Galunsky, Nicolina Stambolieva, Jose Kaneti

Lĩnh vực: Hóa sinh, Khoa học phân tử

Nội dung tài liệu: Nghiên cứu này tập trung vào việc khảo sát động học và mô hình hóa phân tử của enzyme penicillin acylase (PA) từ Escherichia coli. Các thí nghiệm động học với một loạt các dẫn xuất phenylacetyl arylamide đã tiết lộ rằng ít nhất một nhóm phân cực trong phần amin của tiểu đơn vị là cần thiết cho sự định hướng đúng đắn của cơ chất trong vùng liên kết nucleophile lớn của enzyme. Mô hình hóa cơ học lượng tử về tương tác enzyme-cơ chất trong vị trí hoạt động cho thấy rằng, trong trường hợp các cơ chất thiếu đối xứng cục bộ, sự liên kết hiệu quả ngụ ý hai cấu hình không đối xứng tương đối với hai gốc guanidinium mang điện tích dương của ArgA145 và ArgB263. Điều này chỉ ra vai trò then chốt của cặp arginine được chỉ định trong tính đặc hiệu và tính lập thể của penicillin acylase.

Mục lục chi tiết:

• Kinetic studies and molecular modelling attribute a crucial role in the specificity and stereoselectivity of penicillin acylase to the pair ArgA145-ArgB263
• Kinetic experiments with a substrate series of phenylacetyl-arylamides reveal that at least one polar group in the amine moiety is required for the proper orientation of the substrate in the large nucleophile-binding subsite of penicillin acylase of Escherichia coli. Quantum mechanical molecular modelling of enzyme-substrate interactions in the enzyme active site shows that in the case of substrates lacking local symmetry, the productive binding implies two nonsymmetrical arrangements with respect to the two positively charged guanidinium residues of ArgA145 and ArgB263. This indicates a crucial role of the specified arginine pair in the substrate- and stereoselectivity of penicillin acylase.
• Keywords: enzyme kinetics; molecular modelling; nucleophile specificity and stereoselectivity; penicillin acylase.
• Penicillin G acylases (PA, EC 3.5.1.11) from different sources have been widely studied because of their application as industrial biocatalysts for hydrolytic and synthetic transformations in the production of semisynthetic β-lactam antibiotics [1,2] and for their possible new uses in synthetic organic chemistry [3-5]. PA has been identified as an N-terminal nucleophile hydrolase following specific catalytic and processing mechanisms [6-8]. During evolution, the catalytic properties of enzymes have been optimized for their function in vivo. However, their application as industrial biocatalysts often requires transformations of substrates not encountered in nature under reaction conditions differing from physiological ones. Protein design aimed at rational optimization and/or effective screening of enzymes for new transformations requires the study of their specificity with appropriate substrate series. Convenient substrates should also contain sensitive reporter groups for spectrophotometric or fluorimetric detection to facilitate rapid and reliable kinetic measurements.
• The acyl specificity of PA is restricted to aromatic molecules and has been investigated mainly with substrates containing phenylacetyl, phenylglycyl, mandelyl, pyridyl-acetyl or other arylacetyl moieties [9-13]. Our previous studies of PA catalysed transfer reactions with a nonspecific acyl moiety, benzoxazol-2-on-3-yl-acetyl, have shown that the hydrolytic ability of PA for such substrates is drastically decreased, but that its nucleophile specificity is more pronounced and the synthetic capacity is, respectively, increased. In the latter system PA behaves as a typical transferase [14,15]. The nucleophile or S₁’ [16] specificity of the most studied PA from Escherichia coli has been probed in both hydrolytic and kinetically controlled transfer reactions, but the quantitative data published so far are scarce. Specificity constants for PA catalysed hydrolysis of phenylacetyl derivatives with variable leaving groups such as β-lactam nuclei, amino acids, peptides and nucleosides have been shown to differ up to three orders of magnitude [13,17]. Structural, site-directed mutation and kinetic investigations have identified several active site residues important for the S₁’-P₁’ interactions relevant to the catalytic mechanism [18–21]. There are, however, still questions to be answered about the alternatives of interactions in the large nucleophile binding subsite. In the substrate series studied here the phenylacetyl moiety is kept constant and the leaving group structures are confined to arylamines. The expected output is a set of comparative kinetic data, which combined with molecular modelling based on available crystallographic data, could give more detailed information on PA nucleophile binding subsite and the mechanism of transformations with this class of compounds. These data can be used further for rational design of substrates for different purposes, e.g. screening of protein engineered PA for new enzymatic transformations, and analysis of kinetics of ‘invisible’ substrates [22]. The correlation of the kinetic parameters with the nucleophile structure could also allow the design of substrate mimetics for more effective acyl transfer [23].
• Materials and methods
• Materials
• Substrates
• Table 1. Phenylacetyl arylamides used as probes for the nucleophile binding subsite of E. coli PA.
• Kinetic measurements
• Modelling and analysis of interaction energies within the active site
• Results and discussion
• Substrate structure and kinetic results
• Molecular modelling
• Fig. 1. The hydrogen bond network (broken lines) around ArgB263 as derived from X-ray coordinates of amino acid residues in penicillin acylase [19] and AM1 docking calculations. The three N atoms of the side chain 8-guanidino group of this residue are involved in H-bonding as follows: Ne with the O atom of the main chain CO group of the TrpB240; Nη¹ shares H bonding with O atom of the CO group of LeuB387 together with O atom of γ-OH of SerB386; Nn² is in H bonding with Oδ¹ of AsnB241. In the free enzyme, a bridging water molecule W360 bonded with γ-OH of SerB386 and Nn² of ArgB263, respectively, closes the H-bond network. Atom colours used are: C, green; N, blue; O, red; S, yellow.
• Fig. 2. The optimized positions of some arylamide substrates docked in the active site of penicillin acylase by AM1 calculations. Amino acid residues of the enzyme are at their X-ray coordinates [19]. Substrates having local symmetry of the leaving group (top left and bottom right) bind at an equilibrium point of the electrostatic action of the two guanidinium residues. Substrates with leaving groups lacking local symmetry (for NIPAB, bottom left, COO is close to ArgB263; for iso-NIPAB, top right, NO2 is close to ArgA145) can assume either of the two directions depending on the orientation of the polar group.
• Fig. 3. Penicillin acylase stereoselection of achiral arylamide substrate with a leaving group lacking local symmetry. The simultaneous strong electrostatic action of the two positively charged residues, ArgA145 and ArgB263, on the negatively charged COO in the aminic part determines the position of the substrate within the S₁’-binding subsite.
• Table 2. Steady-state kinetic data for PA catalysed hydrolysis of phenylacetyl arylamides with different leaving groups. Reaction conditions: 25 °C, 50 mm phosphate buffer pH 7.0, 10% dimethyl sulfoxide. The SD from the mean value was < 10% in three determinations. Substrate numbering is the same as in Table 1.
• The proper orientation of COOH and NO2 substituents in the aminoaryl moiety favours the hydrolytic reaction of NIPAB. PhAc-arylamides with only one (NO2 or COOH) substituent are substantially worse substrates. The data for the PA catalysed hydrolysis of PhAc-Asp and PhAc-Glu [13] and our data for PhAc-pAB, PhAc-mAB and PhAc-OAB (Table 2) imply that the COOH group has to be positioned as in NIPAB for effective catalysis. The incorporated pyridyl moiety in N-(5-nitro-2-pyridyl)-phenylacetamide (NIPPA) might lead to alternative interactions within the active site, resulting in different orientation and binding.
• Quantum mechanical molecular modelling
• AM1 optimization of substrate position and conformation within our selection of PA active site fragments places NIPAB somewhat closer to ArgB263 than to ArgA145. The distances between the polar CO2 and O(NO) and positively charged guanidinium fragments of ArgA145 and ArgB263 are listed in Table 3. AM1 docking calculations show uniformly that PG and phenylacetyl arylamides have the benzyl fragment PhCH2 placed in the hydrophobic groove of the active site. The polar fragments of all substrates align between the positively charged ArgA145 and ArgB263, with the carboxyl group of PG somewhat closer to ArgA145, while nitroarylamides have the polar NO2 at roughly equal distances from the two guanidinium fragments. More important, the COO groups of arylamides on Figs 2 and 3 are at approximately equal distances from the two positively charged fragments as well, Table 3. The complexes of selected phenylacetyl arylamides with the mentioned selection of amino acid and oligopeptide residues from the active site of PA are shown on Figs 1,2 and 3. The AM1 docked complex of PG is shown on Fig. 1. The discussed hydrogen bond network around ArgB263 is retained also with arylamide substrates and involves their polar carboxylate and/or nitro groups. For substrates with leaving groups, lacking local symmetry, e.g. NIPAB, PG, as well as with the poor substrate PhAc-mAB, we were able to model complexes with the mentioned selection of amino acid residues around the catalytic site of PA, having the COOH group directed to either ArgA145, or ArgB263 (Fig. 3). These results emphasize the caveat of multiple minima for the accommodation of substrate within the active site of PA. More importantly, however, the possibility of polar group orientation towards either ArgA145 or ArgB263 indicates a source of substrate specificity and stereoselectivity of PA at the molecular level. Experimental observations on the pH dependence of PA enantioselectivity [51] coincides well with the above conclusions. Computational modelling of PA enantioselectivity in the reverse reaction of amide bond synthesis also has pointed at the role of ArgB263 in this process [48]. The decomposition of interaction energies within the studied complexes, shown in Table 4, indicates relatively
• Table 3. Distances (Å) between polar substrates groups (CO₂ and NO₂) and the two ArgB263 and ArgA145 residues of penicillin acylase as a result of AM1 optimization of the corresponding substrate position in the selected PA fragment environment.
• Table 4. Calculated STO-3G interaction energies (kcalmol⁻¹) for various substrates with the larger 10-fragment selection of protein residues within the active site of penicillin acylase. Superscripts at total energies indicate the direction of nonsymmetrical polar groups towards either ArgA145 or ArgB263. Partial energy contributions from the two arginine residues and the substrate are given vs. measured Km values given in Table 2.
• smaller contributions of ArgA145 to individual terms of the interactions: electrostatic, charge transfer, and polarization. On the contrary, contributions from ArgB263 are significantly larger and generally comparable to those of the nucleophile SerB1, GlnB23-PheB24, AlaB69, believed to be the dominant substrate binding fragments of PA. In addition, substrates with leaving groups lacking local symmetry may have their polar group directed towards ArgB263. In this case calculated interaction energies with the rest of the complex are larger than in the case of substrates with the polar group oriented towards ArgA145. The interaction energy of ArgA145 itself with the rest of the complex is large when the substrate’s polar group is directed toward it, and small when the polar group points to ArgB263. The interaction energy of ArgB263 with the rest of the complex, however, remains large and practically constant irrespective of the orientation of the polar group. While a significant part of the latter relatively large interaction energy can be attributed to the hydrogen bonding network around ArgB263, the mentioned findings give another argument favouring the importance of ArgB263 in substrate binding to PA. The pronounced difference in interaction energies of the two arginine residues shows certain capability of the pair of polar guanidinium groups to discern between orientations of substrates in the active site. These two arginines should thus contribute significantly to enzyme stereoselectivity. A more detailed account of the Morokuma analysis [39] of interaction energies in PA active site complexes will be given elsewhere (J. Kaneti, S. Bakalova, I. Ivanov, M. Guncheva & N. Stambolieva, unpublished data).
• Conclusions
• Kinetic and molecular modelling studies with a substrate series of phenylacetyl arylamides reveal that at least one polar group in the amine moiety of the substrate is essential for its proper orientation in the large nucleophile binding subsite of penicillin acylase.
• AM1 docking calculations based on the crystal structure [19] give evidence of polar environment around ArgB263. It consists of O atoms of the main chain CO groups of LeuB387 and TrpB240, O8¹ atom of AsnB241 and Oy atom of SerB386 and is expected to stabilize the positive charge of ArgB263.
• The possible nonsymmetrical accommodation of substrates with respect to the pair of ArgA145 and ArgB263 of PA gives rise to notable three-dimensional stereochemical differences in their corresponding enzyme-substrate complexes, and to a certain degree of stereoselectivity. The pair ArgA145 and ArgB263 significantly influences the S specificity and contributes to the appropriate docking of the substrate.
• References