Understanding the enzymatic activity of 4-oxalocrotonate tautomerase and its mutant analogues: a computational study

The effect of replacing arginine residues (Arg) with citrulline residues (Cit) in the binding site of 4-oxalocrotonate tautomerase (4-OT) was investigated with force field molecular dynamics and hybrid quantum mechanics/molecular mechanics studies. It is found that the Arg61Cit mutation has only minor effects on the k(cat) and K(M) values determined experimentally because of the flexibility of this residue. The decrease in k(cat) and increase in K(M) for the Arg11Cit and Arg39Cit mutations are due to the disruption of the binding site, which arises from repulsive interactions with neighboring residues. The results of this investigation shed new light on the effects of mutations in the binding site of 4-OT and consequently on how the enzyme binds the active substrate.     

Electrostatic interactions dominate the catalytic contribution of arg39 in 4-oxalocrotonate tautomerase

The synthesis and kinetic parameters of a comprehensive set of 4-OT analogues with arginine (X = NH2+) to citrulline (X = O) substitutions at positions 11, 39, and 61 are reported. These data suggest that the main contribution of Arg39' ' to catalysis is by electrostatic stabilization of the anionic transition state leading to intermediate 2, and not by hydrogen bonding.     

Selective stabilization of the chorismate mutase transition state by a positively charged hydrogen bond donor

Citrulline was incorporated via chemical semisynthesis at position 90 in the active site of the AroH chorismate mutase from Bacillus subtilis. The wild-type arginine at this position makes hydrogen-bonding interactions with the ether oxygen of chorismate. Replacement of the positively charged guanidinium group with the isosteric but neutral urea has a dramatic effect on the ability of the enzyme to convert chorismate into prephenate. The Arg90Cit variant exhibits a >104-fold decrease in the catalytic rate constant kcat with a 2.7-fold increase in the Michaelis constant Km. In contrast, its affinity for a conformationally constrained inhibitor molecule that effectively mimics the geometry but not the dissociative character of the transition state is only reduced by a factor of approximately 6. These results show that an active site merely complementary to the reactive conformation of chorismate is insufficient for catalysis of the mutase reaction. Instead, electrostatic stabilization of the polarized transition state by provision of a cationic hydrogen bond donor proximal to the oxygen in the breaking C-O bond is essential for high catalytic efficiency.     

All electron quantum chemical calculation of the entire enzyme system confirms a collective catalytic device in the chorismate mutase reaction

To elucidate the catalytic power of enzymes, we analyzed the reaction profile of Claisen rearrangement of Bacillus subtilis chorismate mutase (BsCM) by all electron quantum chemical calculations using the fragment molecular orbital (FMO) method. To the best of our knowledge, this is the first report of ab initio-based quantum chemical calculations of the entire enzyme system, where we provide a detailed analysis of the catalytic factors that accomplish transition-state stabilization (TSS). FMO calculations deliver an ab initio-level estimate of the intermolecular interaction between the substrate and the amino acid residues of the enzyme. To clarify the catalytic role of Arg90, we calculated the reaction profile of the wild-type BsCM as well as Lys90 and Cit90 mutant BsCMs. Structural refinement and the reaction path determination were performed at the ab initio QM/MM level, and FMO calculations were applied to the QM/MM refined structures. Comparison between three types of reactions established two collective catalytic factors in the BsCM reaction: (1) the hydrogen bonds connecting the Glu78-Arg90-substrate cooperatively control the stability of TS relative to the ES complex and (2) the positive charge on Arg90 polarizes the substrate in the TS region to gain more electrostatic stabilization.     

Comparative analysis of binding energy of chymostatin with human cathepsin A and its homologous proteins by molecular orbital calculation

Cathepsin A is a mammalian lysosomal enzyme that catalyzes the hydrolysis of the carboxy-terminal amino acids of polypeptides and also regulates beta-galactosidase and neuraminidase-1 activities through the formation of a multienzymic complex in lysosomes. Human cathepsin A (hCathA), yeast carboxypeptidase (CPY), and wheat carboxypeptidase II (CPW) belong to the alpha/beta-hydrolase fold family. They have structurally similar active-site clefts, but there are small differences in the amino acid residues comprising their active sites that might determine the substrate specificity and sensitivity to microbial inhibitors including chymostatin. To examine the selectivity and binding mechanism of chymostatin as to hCathA, CPY, and CPW at the atomic level, we analyzed the interaction energy between chymostatin and each protein quantitatively by semiempirical molecular orbital calculation AM1 with the continuum solvent model. We predicted the electrostatic repulsion between the P3 cyclic arginine residue of the inhibitor and the Arg344 in the S3 active subsite of hCathA. Genetic conversion of Arg344 of the wild-type hCathA to Ile also caused an increase in its sensitivity to chymostatin, which was correlated with the decrease in the interaction energy calculated with the molecular orbital method. The present results suggest that such molecular calculation should be useful for evaluating the interactions between ligands, including inhibitors and homologous enzymes, in their docking models.     

Redox control of the P450cam catalytic cycle: effects of Y96F active site mutation and binding of a non-natural substrate

Spectroelectrochemistry measurements are used to demonstrate that active site mutation and binding of an non-natural substrate to P450cam (CYP101) reduces the shift in the redox potential caused by substrate-binding, and thereby results in slower catalytic turnover rate relative to wild-type enzyme with the natural camphor substrate.     

Enzymatic Modification of Soluble Cyanophycin Using the Type II Peptidyl Arginine Deiminase from Oryctolagus cuniculus

An increased structural variety expands the number of putative applications for cyanophycin (multi‐l ‐arginyl‐poly‐[l ‐aspartic acid], CGP). Therefore, structural modifications of CGP are of major interest; these are commonly obtained by modification and optimization of the bacterial producing strain or by chemical modification. In this study, an enzymatic modification of arginine side chains from lysine‐rich CGP is demonstrated using the peptidyl arginine deiminase from Oryctolagus cuniculus, purified from Escherichia coli after heterologous expression. About 10% of the arginine side chains are converted to citrulline which corresponds to 4% of the polymer's total side chains. An inhibition of the reaction in the presence of small amounts of l ‐citrulline is observed, thereby explaining the low conversion rate. CGP dipeptides can be modified with about 7.5 mol% of the Asp‐Arg dipeptides being converted to Asp‐Cit. These results show that the enzymatic modification of CGP is feasible, opening up a whole new area of possible CGP modifications for further research.

     

Differential transition-state stabilization in enzyme catalysis: quantum chemical analysis of interactions in the chorismate mutase reaction and prediction of the optimal catalytic field

Chorismate mutase is a key model system in the development of theories of enzyme catalysis. To analyze the physical nature of catalytic interactions within the enzyme active site and to estimate the stabilization of the transition state (TS) relative to the substrate (differential transition state stabilization, DTSS), we have carried out nonempirical variation-perturbation analysis of the electrostatic, exchange, delocalization, and correlation interactions of the enzyme-bound substrate and transition-state structures derived from ab initio QM/MM modeling of Bacillus subtilis chorismate mutase. Significant TS stabilization by approximately -23 kcal/mol [MP2/6-31G(d)] relative to the bound substrate is in agreement with that of previous QM/MM modeling and contrasts with suggestions that catalysis by this enzyme arises purely from conformational selection effects. The most important contributions to DTSS come from the residues, Arg90, Arg7, Glu78, a crystallographic water molecule, Arg116, and Arg63, and are dominated by electrostatic effects. Analysis of the differential electrostatic potential of the TS and substrate allows calculation of the catalytic field, predicting the optimal location of charged groups to achieve maximal DTSS. Comparison with the active site of the enzyme from those of several species shows that the positions of charged active site residues correspond closely to the optimal catalytic field, showing that the enzyme has evolved specifically to stabilize the TS relative to the substrate.     

Ab initio QM/MM study shows there is no general acid in the reaction catalyzed by 4-oxalocrotonate tautomerase

The mechanism for the reaction catalyzed by the 4-oxalocrotonate tautomerase (4-OT) enzyme has been studied using a quantum mechanical/molecular mechanical (QM/MM) method developed in our laboratory. Total free energy barriers were obtained for the two steps involved in this reaction. In the first step, Pro-1 acts as a general base to abstract a proton from the third carbon of the substrate, 2-oxo-4-hexenedioate, creating a negative charge on the oxygen at C-2 of this substrate. In the second step, the same hydrogen abstracted by the N-terminal Pro-1 is shuttled back to the fifth carbon of the substrate to form the product, 2-oxo-3-hexenedioate. The calculated total free energy barriers are 14.54 and 16.45 kcal/mol for the first and second steps, respectively. Our calculations clearly show that there is no general acid in the reaction. Arg-39' ', which is hydrogen bonded to the carboxylate group of the substrate, and an ordered water, which moves closer to the site of the charge formed in the transition state and intermediate, play the main role in transition state/intermediate stabilization without acting as general acids in the reaction.     

What is so special about Arg 55 in the catalysis of cyclophilin A? insights from hybrid QM/MM simulations

Potential of mean force (PMF) simulations with a hybrid QM/MM potential function were used to analyze the catalytic mechanism of human cyclophilin A (CypA). PMF calculations were performed for proline isomerization of peptides in solution, the wild-type CypA, and several CypA mutants. With an approximate density functional theory, the self-consistent-charge density functional tight binding (SCC-DFTB) as the QM level, and CHARMM 22 force field as MM, satisfactory energetics compared to available experiments were obtained. Calculations for the Arg55Ala and zero-charge-Arg55 mutants clearly indicated that Arg 55 significantly stabilizes the isomerization transition state through electrostatic interactions. However, the decrease in the average distance (thus the increase in interaction) between Arg 55 and the substrate amide N in going from the stable states to the transition state is mainly due to the pyramidalization of the amide N rather than motions associated with Arg 55. Although the nanosecond simulations cannot exclude the existence of sub-millisecond collective motions proposed on the basis of recent elegant NMR relaxation and line-shape analyses, the energetics obtained for the various enzyme systems here indicate that the contribution from motions of active site residues to catalysis is expected to be small. Instead, the present simulations support that the structural stability rather than mobility of the preorganized active site is more important. Through hydrogen-bonding interactions among the substrate, Arg 55, Gln 63, and Asn 102, the active site of the wild-type enzyme is structurally very stable and puts Arg 55 in a favorable position to perform its catalytic role in the transition state. This is further illustrated with the somewhat unexpected prediction that Arg55Lys is largely catalytically inactive, because Lys does not have the unique bifurcating construct of the guanidino group in Arg and thus the active site of Arg55Lys cannot accommodate Lys in a position capable of providing electrostatic stabilization of the isomerization transition state. Among all the enzyme systems studied, the wild-type CypA is the only one that selects the syn/exo transition state, while the syn/endo conformation is also present in the mutants, which is another reason for their higher barriers. Finally, the present analysis indicated that the population of near-attack-conformations (NAC) is not relevant to catalysis in CypA.     

Substrate orientation in 4-oxalocrotonate tautomerase and its effect on QM/MM energy profiles

The tautomerization of 2-oxo-4E-hexendioate by 4-oxalocrotonate tautomerase has been studied by quantum mechanical/molecular mechanical (QM/MM) methods using three models, A-C, with different substrate orientations. The computed QM/MM energy profiles are rather different. Various energy partitioning analyses indicate the origin of these differences and the role of the active site residues for different substrate orientations. The proposed new model C is preferred over the previously used models A and B because it combines favorable substrate binding geometries with reasonable barriers and is consistent with the experimental evidence from mutation studies concerning the catalytic ability of specific residues in the binding site, especially R11'.     

The 4-oxalocrotonate tautomerase- and YwhB-catalyzed hydration of 3E-haloacrylates: implications for the evolution of new enzymatic activities

4-Oxalocrotonate tautomerase (4-OT) catalyzes the conversion of 2-oxo-4E-hexenedioate to 2-oxo-3E-hexenedioate through the intermediate, 2-hydroxy-2,4E-hexadienedioate. 4-OT and a homologue found in Bacillus subtilis (designated YwhB) share sequence identity and two key catalytic groups, Pro-1 and Arg-11, with the two subunits comprising trans-3-chloroacrylic acid dehalogenase (CaaD). 4-OT and YwhB have now been found to display a low-level hydratase activity, resulting in the dehalogenation of 3E-haloacrylates. The enzymes are highly selective for the (E)-isomer, and Pro-1 is critical for the activity while an arginine is likely required. Two mechanisms are proposed in which Pro-1 functions as a general base or a general acid catalyst and, along with the arginine, facilitates the Michael addition of water. Both mechanisms suggest an intriguing route for the evolution of the CaaD activity. One or more mutations could decrease the hydrophobic environment of the active site, which would make it more favorable for a hydrolytic reaction, thereby raising the pKa of Pro-1 and increasing the concentration of enzyme in the reactive form.     

Enhancing the Peroxygenase Activity of a Cofactor-Independent Peroxyzyme by Directed Evolution Enabling Gram-Scale Epoxide Synthesis

Peroxygenases selectively incorporate oxygen into organic molecules making use of the environmentally friendly oxidant H₂O₂ with water being the sole by-product. These biocatalysts can provide ‘green’ routes for the synthesis of enantioenriched epoxides, which are fundamental intermediates in the production of pharmaceuticals. The peroxyzyme 4-oxalocrotonate tautomerase (4-OT), catalysing the epoxidation of a variety of α,β-unsaturated aldehydes with H₂O₂, is outstanding because of its independence from any cost-intensive cofactor. However, its low-level peroxygenase activity and the decrease in the enantiomeric excess of the corresponding α,β-epoxy-aldehydes under preparative-scale conditions is limiting the potential of 4-OT. Herein we report the directed evolution of a tandem-fused 4-OT variant, which showed an ∼150-fold enhanced peroxygenase activity compared to 4-OT wild type, enabling the synthesis of α,β-epoxy-aldehydes in milligram- and gram-scale with high enantiopurity (up to 98 % ee) and excellent conversions. This engineered cofactor-independent peroxyzyme can provide new opportunities for the eco-friendly and practical synthesis of enantioenriched epoxides at large scale.     

Physical nature of interactions within the active site of cytosine-5-methyltransferase

The physical nature of interactions within the active site of cytosine-5-methyltransferase (CMT) was studied using a variation-perturbation energy decomposition scheme defining a sequence of approximate intermolecular interaction energy models. These models have been used to analyze the catalytic activity of residues constituting cytosine-5-methyltransferase active site as well their role in the binding group of de novo designed inhibitors. Our results indicate that Glu119, Arg163, and Arg165 appear to play the dominant role in stabilizing the protonated transition state structure and their influence can be qualitatively approximated by electrostatic interactions alone. The stabilization of neutral structures of the alternative reaction pathway is small, which might suggest the protonated pathway as preferred by the enzyme. Exchange and delocalization terms are negligible in most cases, or they cancel each other to some extent. Interactions of inhibitors with the CMT active site are dominated by electrostatic multipole contributions in analogy with previously studied transition state analogue inhibitors of leucyl aminopeptidase.     

A practical synthesis of zanamivir phosphonate congeners with potent anti-influenza activity

Two phosphonate compounds 1a (4-amino-1-phosphono-DANA) and 1b (phosphono-zanamivir) are synthesized and shown more potent than zanamivir against the neuraminidases of avian and human influenza viruses, including the oseltamivir-resistant strains. For the first time, the practical synthesis of these phosphonate compounds is realized by conversion of sialic acid to peracetylated phosphono-DANA diethyl ester (5) as a key intermediate in three steps by a novel approach. In comparison with zanamivir, the high affinity of 1a and 1b can be partly attributable to the strong electrostatic interactions of their phosphonate groups with the three arginine residues (Arg118, Arg292, and Arg371) in the active site of neuraminidases. These phosphonates are nontoxic to the human 293T cells; they protect cells from influenza virus infection with EC(50) values in low-nanomolar range, including the wild-type WSN (H1N1), the 2009 pandemic (H1N1), the oseltamivir-resistant H274Y (H1N1), RG14 (H5N1), and Udorn (H3N2) influenza strains.     

Proton delivery to ferryl heme in a heme peroxidase: enzymatic use of the Grotthuss mechanism

We test the hypothesized pathway by which protons are passed from the substrate, ascorbate, to the ferryl oxygen in the heme enzyme ascorbate peroxidase (APX). The role of amino acid side chains and bound solvent is demonstrated. We investigated solvent kinetic isotope effects (SKIE) for the wild-type enzyme and several site-directed replacements of the key residues which form the proposed proton path. Kinetic constants for H(2)O(2)-dependent enzyme oxidation to Compound I, k(1), and subsequent reduction of Compound II, k(3), were determined in steady-state assays by variation of both H(2)O(2) and ascorbate concentrations. A high value of the SKIE for wild type APX ((D)k(3) = 4.9) as well as a clear nonlinear dependence on the deuterium composition of the solvent in proton inventory experiments suggest the simultaneous participation of several protons in the transition state for proton transfer. The full SKIE and the proton inventory data were modeled by applying Gross-Butler-Swain-Kresge theory to a proton path inferred from the known structure of APX. The model has been tested by constructing and determining the X-ray structures of the R38K and R38A variants and accounts for their observed SKIEs. This work confirms APX uses two arginine residues in the proton path. Thus, Arg38 and Arg172 have dual roles, both in the formation of the ferryl species and binding of ascorbate respectively and to facilitate proton transfer between the two.     

Mapping tertiary interactions in protein folding reactions: a novel mass spectrometry- and chemical synthesis-based approach

A novel mass spectrometry- and chemical synthesis-based approach for studying protein folding reactions is described, and its initial application to study the folding/unfolding reaction of a homo-hexameric enzyme 4-oxalocrotonate (4OT) is reported. This new approach involves the application of total chemical synthesis to prepare protein analogues that contain a photoreactive amino acid site-specifically incorporated into their primary amino acid sequence. To this end, a photoreactive amino acid-containing analogue of 4OT in which Pro-1 was replaced with p-benzoyl-l-phenylalanine (Bpa) was prepared. This analogue can be used to map structurally specific protein-protein interactions in 4OT's native folded state. These photocrosslinking studies and peptide mapping results with (PlBpa)4OT indicate that this construct is potentially useful for probing the structural properties of equilibrium and kinetic intermediates in 4OT's folding reaction.     

Quantum Mechanics/Molecular Mechanics Studies on the Mechanism of Action of Cofactor Pyridoxal 5′‐Phosphate in Ornithine 4,5‐Aminomutase

A computational study was performed on the experimentally elusive cyclisation step in the cofactor pyridoxal 5′‐phosphate (PLP)‐dependent D ‐ornithine 4,5‐aminomutase (OAM)‐catalysed reaction. Calculations using both model systems and a combined quantum mechanics/molecular mechanics approach suggest that regulation of the cyclic radical intermediate is achieved through the synergy of the intrinsic catalytic power of cofactor PLP and the active site of the enzyme. The captodative effect of PLP is balanced by an enzyme active site that controls the deprotonation of both the pyridine nitrogen atom (N1) and the Schiff‐base nitrogen atom (N2). Furthermore, electrostatic interactions between the terminal carboxylate and amino groups of the substrate and Arg297 and Glu81 impose substantial “strain” energy on the orientation of the cyclic intermediate to control its trajectory. In addition the “strain” energy, which appears to be sensitive to both the number of carbon atoms in the substrate/analogue and the position of the radical intermediates, may play a key role in controlling the transition of the enzyme from the closed to the open state. Our results provide new insights into several aspects of the radical mechanism in aminomutase catalysis and broaden our understanding of cofactor PLP‐dependent reactions.     

Mapping tertiary interactions in protein folding reactions: a novel mass spectrometry- and chemical synthesis-based approach

A novel mass spectrometry- and chemical synthesis-based approach for studying protein folding reactions is described, and its initial application to study the folding/unfolding reaction of a homo-hexameric enzyme 4-oxalocrotonate (4OT) is reported. This new approach involves the application of total chemical synthesis to prepare protein analogues that contain a photoreactive amino acid site-specifically incorporated into their primary amino acid sequence. To this end, a photoreactive amino acid-containing analogue of 4OT in which Pro-1 was replaced with p-benzoyl-l-phenylalanine (Bpa) was prepared. This analogue can be used to map structurally specific protein-protein interactions in 4OT’s native folded state. These photocrosslinking studies and peptide mapping results with (P1Bpa)4OT indicate that this construct is potentially useful for probing the structural properties of equilibrium and kinetic intermediates in 4OT’s folding reaction.