Molecular orbital studies of enzyme activity: I: Charge relay system and tetrahedral intermediate in acylation of serine proteinases (original) (raw)
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Journal of Molecular Structure-theochem, 1982
The catalytic triad of serine proteinases is composed of serine, histidine and aspartate residues. During catalysis the imidazole group of histidine, as a general base, accepts the proton from the hydroxyl group of serine. In this work the equilibrium states of the proton between the protonated imidazole group and the aspartate ion, as well as the factors determining this equilibrium, are examined by various quantum chemical approximations. The protonated histidineaspartate diad of subtilisin BPN' has been modelled by the imidazole-formic acid system. It is found that the CNDO/I and ab initio STO-3G basis set calculations overestimate the stability of the neutral form where the proton is attached to the aspartate side chain. A more probable proton transfer energy is obtained if the total energy of the diad is partitioned into bond and interaction energy terms calculated with 4-31G and STO-3G basis sets, respectively. However, even this approximation is insufficiently accurate to establish unambiguously the position of the proton in the gas phase. Nevertheless, the effects cf the environment on proton transfer could be estimated with more certainty. It is found that each component of the environment, i.e. (1) aminoacid residues of subtilisin within 1 nm of the diad, (2) three water molecules and (3) a counter ion, stabilizes the imidazolium-aspartate ion pair of the diad so that the proton cannot be transferred from the imidazolium to the aspartate ion. These results clearly demo&ate the crucial role of the environment in the reactivities of the catalytic group of enzymes.
Journal of Computational Chemistry, 1992
Ab initio and semiempirical (AMl) molecular orbital theory has been used to model the cleavage of formamide at the active site of carboxypeptidase A. The model active site consists of a zinc dication coordinated to two imidazoles, an acetate, a water with a hydrogen-bonded formate, and a formamide molecule as model substrate. AM1 has been compared with ab initio theory for the coordination of water and formamide to Zn++ and found to give excellent energetic results. The course of the amide cleavage was therefore calculated with AM1. The first step of the reaction is the dissociation of the zinc-coordinated water to give an active ZnOH+ species. The remote formate acts as proton acceptor. This process has an activation energy of only 4.6 kcal mol-'. The next and rate-determining step is the concerted addition of the ZnOH+ moiety to the formamide C=O bond. The Zn-0 distance in the transition state is more than 3 A. In four further steps, the amide nitrogen is protonated and the C-N bond cleaved. The net activation energy for the entire process is 15.5 kcal mol-' relative to the active site model and 19.6 kcal mol-' relative to the most stable point on the calculated reaction profile. 'Author to whom all correspondence should be addressed. and Lipscomb6 reported an MO study on the hydrolysis of peptides by CPA. Osman and Weinstein7 examined models for active sites of metalloenzymes and compared zinc-and beryllium-containing complexes by LCAO-SCF calculations, and Christianson and Alexander8 studied the role played by carboxylate-histidine-zinc interactions in protein structures and function. Pardo et al.9 theoretically examined models for proton transfer in biological systems. However, none of these publications proposed a full reaction path for the cleavage of peptides by CPA in an active-site model. This article reports a combined ab initio and AM1 study of the mechanism of peptide cleavage by CPA. We used an active-site model similar to that used by Merz et al. and find significant parallels to their calculated mode of activity for carbonic anhydrase. Of the many zinc peptidases, thermolysin (Tln) and CPA are the best characterized. In both cases, X-ray structures of enzymehnhibitor complexes are available. These structures formed the basis for our active site model. CPA catalyzes the hydrolysis of C-terminal amino acids and shows a strong preference for cleaving C-terminal residues that contain aromatic or branched chains. l2 Three realistic mechanisms have been proposed to explain the action of CPA: the anhydride mechanism,
Enzyme:Substrate Hydrogen Bond Shortening during the Acylation Phase of Serine Protease Catalysis
Biochemistry, 2006
Atomic resolution (e1.2 Å) serine protease intermediate structures revealed that the strength of the hydrogen bonds between the enzyme and the substrate changed during catalysis. The well-conserved hydrogen bonds of antiparallel -sheet between the enzyme and the substrate become significantly shorter in the transition from a Michaelis complex analogue (Pontastacus leptodactylus (narrow-fingered crayfish) trypsin (CFT) in complex with Schistocerca gregaria (desert locust) trypsin inhibitor (SGTI) at 1.2 Å resolution) to an acyl-enzyme intermediate (N-acetyl-Asn-Pro-Ile acyl-enzyme intermediate of porcine pancreatic elastase at 0.95 Å resolution) presumably synchronously with the nucleophilic attack on the carbonyl carbon atom of the scissile peptide bond. This is interpreted as an active mechanism that utilizes the energy released from the stronger hydrogen bonds to overcome the energetic barrier of the nucleophilic attack by the hydroxyl group of the catalytic serine. In the CFT:SGTI complex this hydrogen bond shortening may be hindered by the 27I-32I disulfide bridge and Asn-15I of SGTI. The position of the catalytic histidine changes slightly as it adapts to the different nucleophilic attacker during the transition from the Michaelis complex to the acyl-enzyme state, and simultaneously its interaction with Asp-102 and Ser-214 becomes stronger. The oxyanion hole hydrogen bonds provide additional stabilization for acyl-ester bond in the acyl-enzyme than for scissile peptide bond of the Michaelis complex. Significant deviation from planarity is not observed in the reactive bonds of either the Michaelis complex or the acyl-enzyme. In the Michaelis complex the electron distribution of the carbonyl bond is distorted toward the oxygen atom compared to other peptide bonds in the structure, which indicates the polarization effect of the oxyanion hole.
Journal of Theoretical Biology, 1985
The proton relay system of a-chymotrypsin is analyzed by the INDO-ISCRF method. The effects of the protein electric and polarization fields are explicitly introduced in the calculations. It is shown that the multicharged structure Set-His+Asp -is the most sensitive, from an energetic view-point, towards the protein surrounding effects. Variations in the permanent and polarization fields are discussed, as well as the influence of the substrate and one water molecule localized in the active site of the enzyme. The catalytic role of such changes is conjectured. t
2011
Quantum mechanical/molecular mechanical (QM/MM) free energy simulations are applied for understanding the mechanism of the acylation reaction catalyzed by sedolisin, a representative serine-carboxyl peptidase, leading to the acyl-enzyme (AE) and first product from the enzymecatalyzed reaction. One of the interesting questions to be addressed in this work is the origin of the substrate specificity of sedolisin that shows a relatively high activity on the substrates with Glu at P 1 site. It is shown that the bond making and breaking events of the acylation reaction involving a peptide substrate (LLE*FL) seem to be accompanied by local conformational changes, proton transfers as well as the formation of alternative hydrogen bonds. The results of the simulations indicate that the conformational change of Glu at P 1 site and its formation of a low barrier hydrogen bond with Asp-170 (along with the transient proton transfer) during the acylation reaction might play a role in the relatively high specificity for the substrate with Glu at P 1 site. The role of some key residues in the catalysis is confirmed through free energy simulations. Glu-80 is found to act as a general base to accept a proton from Ser-287 during the nucleophilic attack and then as a general acid to protonate the leaving group (N-H of P 1 0-Phe) during the cleavage of the scissile peptide bond. Another acidic residue, Asp-170, acts as a general acid catalyst to protonate the carbonyl of P 1-Glu during the formation of the tetrahedral intermediate and as a general base for the formation of the acyl-enzyme. The energetic results from the free energy simulations support the importance of proton transfer from Asp-170 to the carbonyl of P 1-Glu in the stabilization of the tetrahedral intermediate and the formation of a low-barrier hydrogen bond between the carboxyl group of P 1-Glu and Asp-170 in the lowering of the free energy barrier for the cleavage of the peptide bond. Detailed analyses of the proton transfers during acylation are also given.
Theoretical calculations on the acidity of the active site in aspartic proteinases
Biochemistry, 1988
Semiempirical minimal neglect of differential overlapself-consistent field calculations, corrected and modified for multiple hydrogen-bonding interactions, were applied to models of the active site of aspartic proteinases (AP). The propensities of the two active-site aspartates to ionize were compared under the influence of various neighboring residues and of water molecules. Asp-32 and Asp-21 5 in three aspartic proteinases (endothiapepsin, Rhizopus pepsin, and penicillopepsin) are found to be basically asymmetric, ' The numbers of residues in the various AP are according to the Brookhaven protein bank files for endothiapepsin (4APE), Rhizopus pepsin (ZAPR), and penicillopepsin (ZAPP)
Journal of the American Chemical Society, 2005
Sedolisins (serine-carboxyl peptidases) belong to a recently characterized family of proteolytic enzymes (MEROPS S53) that have a fold resembling that of subtilisin and a maximal activity at low pH. 1 This family includes the peptidase CLN2, 2 a human enzyme for which mutations in the encoding CLN2 gene lead to a fatal neurodegenerative disease, classical late-infantile neuronal ceroid lipofuscinosis. The defining features of the sedolisin family are a unique catalytic triad, 4,5 Ser-Glu-Asp (Ser278-Glu78-Asp82 for kumamolisin-As; see ), as well as the presence of an aspartic acid residue (Asp164 for kumamolisin-As) that replaces Asn155 of subtilisin, a residue that creates the oxyanion hole. The X-ray crystallographic and mutagenesis studies 4,5 demonstrated that the serine residue is the catalytic nucleophile, while the nearby Glu is likely to act as the general base to accept the proton from Ser and assist in the nucleophilic attack. A fundamental question for serine-carboxyl peptidases is whether these enzymes use the catalytic mechanism similar to that of classical serine proteases with simple replacements of certain catalytic residues so that they could be active at low pH. Here we demonstrate from quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) simulations that this may not be the case. Unlike serine proteases that use the oxyanion-hole interactions to achieve the electrostatic stabilization of the tetrahedral intermediate and adduct, the members of the sedolisin family seem to stabilize the tetrahedral intermediate and adduct primarily through a general acid-base mechanism (i.e., similar to the mechanism proposed for aspartic proteases 6 ).
Proteins: Structure, Function, and Genetics, 2002
Despite the availability of many experimental data and some modeling studies, questions remain as to the precise mechanism of the serine proteases. Here we report molecular dynamics simulations on the acyl-enzyme complex and the tetrahedral intermediate during the deacylation step in elastase catalyzed hydrolysis of a simple peptide. The models are based on recent crystallographic data for an acylenzyme intermediate at pH 5 and a time-resolved study on the deacylation step. Simulations were carried out on the acyl enzyme complex with His-57 in protonated (as for the pH 5 crystallographic work) and deprotonated forms. In both cases, a water molecule that could provide the nucleophilic hydroxide ion to attack the ester carbonyl was located between the imidazole ring of His-57 and the carbonyl carbon, close to the hydrolytic position assigned in the crystal structure. In the "neutral pH" simulations of the acylenzyme complex, the hydrolytic water oxygen was hydrogen bonded to the imidazole ring and the side chain of Arg-61. Alternative stable locations for water in the active site were also observed. Movement of the His-57 side-chain from that observed in the crystal structure allowed more solvent waters to enter the active site, suggesting that an alternative hydrolytic process directly involving two water molecules may be possible. At the acyl-enzyme stage, the ester carbonyl was found to flip easily in and out of the oxyanion hole. In contrast, simulations on the tetrahedral intermediate showed no significant movement of His-57 and the ester carbonyl was constantly located in the oxyanion hole. A comparison between the simulated tetrahedral intermediate and a time-resolved crystallographic structure assigned as predominantly reflecting the tetrahedral intermediate suggests that the experimental structure may not precisely represent an optimal arrangement for catalysis in solution. Movement of loop residues 216-223 and P 3 residue, seen both in the tetrahedral simulation and the experimental analysis, could be related to product release. Furthermore, an analysis of the geometric data obtained from the simulations and the pH 5 crystal structure of the acyl-enzyme suggests that since His-57 is protonated, in some aspects, this crystal structure resembles the tetrahedral intermediate. Proteins 2002; 47:357-369.
Biochemistry, 1999
Eglin c, turkey ovomucoid third domain, and bovine pancreatic trypsin inhibitor (Kunitz) are all standard mechanism, canonical protein inhibitors of serine proteinases. Each of the three belongs to a different inhibitor family. Therefore, all three have the same canonical conformation in their combining loops but differ in their scaffoldings. Eglin c (Leu 45 at P 1 ) binds to chymotrypsin much better than its Ala 45 variant (the difference in standard free energy changes on binding is -5.00 kcal/mol). Similarly, turkey ovomucoid third domain (Leu 18 at P 1 ) binds to chymotrypsin much better than its Ala 18 variant (the difference in standard free energy changes on binding is -4.70 kcal/mol). As these two differences are within the (400 cal/mol bandwidth (expected from the experimental error), one can conclude that the system is additive. On the basis that isoenergetic is isostructural, we expect that within both the P 1 Ala pair and the P 1 Leu pair, the conformation of the inhibitor's P 1 side chain and of the enzyme's specificity pocket will be identical. This is confirmed, within the experimental error, by the available X-ray structures of complexes of bovine chymotrypsin AR with eglin c (lacb) and with turkey ovomucoid third domain (1cho). A comparison can also be made between the structures of P 1 (Lys + ) 15 of bovine pancreatic trypsin inhibitor (Kunitz) (1mtn and 1cbw) and of the P 1 (Lys + ) 18 variant of turkey ovomucoid third domain (1hja), both interacting with chymotrypsin. In this case, the conformation of the side chains is strikingly different. Bovine pancreatic trypsin inhibitor with (Lys + ) 15 at P 1 binds to chymotrypsin more strongly than its Ala 15 variant (the difference in standard free energy changes on binding is -1.90 kcal/mol). In contrast, turkey ovomucoid third domain variant with (Lys + ) 18 at P 1 binds to chymotrypsin less strongly than its Ala 18 variant (the difference in standard free energies of association is 0.95 kcal/mol). In this case, P 1 Lys + is neither isostructural nor isoenergetic. Thus, a thermodynamic criterion for whether the conformation of a P 1 side chain in the complex matches that of an already determined one is at hand. Such a criterion may be useful in reducing the number of required X-ray crystallographic structure determinations. More importantly, the criterion can be applied to situations where direct determination of the structure is extremely difficult. Here, we apply it to determine the conformation of the Lys + side chain in the transition state complex of a substrate with chymotrypsin. On the basis of k cat /K M measurements, the difference in free energies of activation for Suc-AAPX-pna when X is Lys + and X is Ala is 1.29 kcal/mol. This is in good agreement with the corresponding difference for turkey ovomucoid third domain variants but in sharp contrast to the bovine pancreatic trypsin inhibitor (Kunitz) data. Therefore, we expect that in the transition state complex of this substrate with chymotrypsin, the P 1 Lys + side chain is deeply inserted into the enzyme's specificity pocket as it is in the (Lys + ) 18 turkey ovomucoid third domain complex with chymotrypsin. .