X-ray snapshots of serine protease catalysis reveal a tetrahedral intermediate

Studies on the catalytic mechanism and inhibition of serine proteases are widely used as paradigms for teaching enzyme catalysis. Ground-breaking work on the structures of chymotrypsin and subtilisin led to the idea of a conserved catalytic triad formed by the active site Ser, His and Asp residues. An oxyanion hole, consisting of the peptide amide of the active site serine and a neighbouring glycine, was identified, and hydrogen bonding in the oxyanion hole was suggested to stabilize the two proposed tetrahedral intermediates on the catalytic pathway. Here we show electron density changes consistent with the formation of a tetrahedral intermediate during the hydrolysis of an acyl-enzyme complex formed between a natural heptapeptide and elastase. No electron density for an enzyme-product complex was observed. The structures also suggest a mechanism for the synchronization of hydrolysis and peptide release triggered by the conversion of the sp2 hybridized carbonyl carbon to an sp3 carbon in the tetrahedral intermediate. This affects the location of the peptide in the active site cleft, triggering the collapse of a hydrogen bonding network between the peptide and the beta-sheet of the active site.     

Enzyme:substrate hydrogen bond shortening during the acylation phase of serine protease catalysis

Atomic resolution (相似文献   

Molecular dynamics simulations of the acyl-enzyme and the tetrahedral intermediate in the deacylation step of serine proteases

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 acyl-enzyme 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 acyl-enzyme 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 P3 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.     

Molecular orbital study of the hydroxylation of benzene and monofluorobenzene catalysed by iron-oxo porphyrin π cation radical complexes

 The reaction mechanism for the hydroxylation of benzene and monofluorobenzene, catalysed by a ferryl-oxo porphyrin cation radical complex (compound) is described by electronic structure calculations in local spin density approximation. The active site of the enzyme is modelled as a six-coordinated (Por⁺)Fe(IV)O a_2u complex with imidazole or H₃CS^– as the axial ligand. The substrates under study are benzene and fluorobenzene, with the site of attack in para, meta and ortho position with respect to F. Two reaction pathways are investigated, with direct oxygen attack leading to a tetrahedral intermediate and arene oxide formation as a primary reaction step. The calculations show that the arene oxide pathway is distinctly less probable, that hydroxylation by an H₃CS^––coordinated complex is energetically favoured compared with imidazole, and that the para position with respect to F is the preferred site for hydroxylation. A partial electron transfer from the substrate to the porphyrin during the reaction is obtained in all cases. The resulting charge distribution and spin density of the substrates reveal the transition state as a combination of a cation and a radical σ-adduct intermediate with slightly more radical character in the case of H₃CS^– as axial ligand. A detailed analysis of the orbital interactions along the reaction pathway yields basically different mechanisms for the modes of substrate–porphyrin electron transfer and rupture of the Fe–O bond. In the imidazole-coordinated complex an antibonding π*(Fe–O) orbital is populated, whereas in the H₃CS^––coordinated system a shift of electron density occurs from the Fe–O bond region into the Fe–S bond. Received: 1 July 1995 / Accepted: 18 December 1995     

Impact of cis-proline analogs on peptide conformation

The beta-turn is a common motif in both proteins and peptides and often a recognition site in protein interactions. A beta-turn of four sequential residues reverses the direction of the peptide chain and is classified by the phi and psi backbone torsional angles of residues i + 1 and i + 2. The type VI turn usually contains a proline with a cis-amide bond at residue i + 2. Cis-proline analogs that constrain the peptide to adopt a type VI turn led to peptidomimetics with enhanced activity or metabolic stability. To compare the impact of different analogs on amide cis-trans isomerism and peptide conformation, the conformational preference for the cis-amide bond and the type VI turn was investigated at the MP2/6-31+G** level of theory in water (polarizable continuum water model). Analogs stabilize the cis-amide conformations through different mechanisms: (1) 5-alkylproline, with bulky hydrocarbon substituent on the C(delta) of proline, increases the cis-amide population through steric hindrance between the alkyl substituent and the N-terminal residues; (2) oxaproline or thioproline, the oxazolidine- or thiazolidine-derived proline analog, favors interactions between the dipole of the heterocyclic ring and the preceding carbonyl oxygen; and (3) azaproline, containing a nitrogen atom in place of the C(alpha) of proline, prefers the cis-amide bond by lone-pair repulsion between the alpha-nitrogen and the preceding carbonyl oxygen. Preference for the cis conformation was augmented by combining different modifications within a single proline. Azaproline and its derivatives are most effective in stabilizing cis-amide bonds without introducing additional steric bulk to compromise receptor interactions.     

Structural insights into the roles of water and the 2' hydroxyl of the P site tRNA in the peptidyl transferase reaction

Peptide bond formation is catalyzed at the peptidyl transferase center (PTC) of the large ribosomal subunit. Crystal structures of the large ribosomal subunit of Haloarcula marismortui (Hma) complexed with several analogs that represent either the substrates or the transition state intermediate of the peptidyl transferase reaction show that this reaction proceeds through a tetrahedral intermediate with S chirality. The oxyanion of the tetrahedral intermediate interacts with a water molecule that is positioned by nucleotides A2637 (E. coli numbering, 2602) and (methyl)U2619(2584). There are no Mg2+ ions or monovalent metal ions observed in the PTC that could directly promote catalysis. The A76 2' hydroxyl of the peptidyl-tRNA is hydrogen bonded to the alpha-amino group and could facilitate peptide bond formation by substrate positioning and by acting as a proton shuttle between the alpha-amino group and the A76 3' hydroxyl of the peptidyl-tRNA.     

Evidence for a tetrahedral intermediate complex during serpin-proteinase interactions

Proteinase inhibitors in the serpin family form complexes with serine proteinases by interactions between the gamma-OH group at serine 195 of the enzyme and a specific peptide bond within the reactive site loop of the inhibitor. However, the type of complex formed (i.e. Michaelis, acyl, or tetrahedral) is unknown. Until now, 13C NMR spectroscopy studies have only been useful in examining complexes formed with either peptide-related or small protein inhibitors, where 13C-labeled amino acids can be inserted semi-synthetically. Recombinant DNA technology has, however, made it possible to specifically enrich larger proteins with 13C. In the case of serpins we have examined the structure of the complex formed between human alpha 1-proteinase inhibitor uniformally labeled with [13C]methionine and porcine pancreatic elastase. 13C NMR spectroscopic studies revealed a large upfield chemical shift of the carbonyl signal of Met-358 upon complex formation suggesting for the first time that a tetrahedral adduct is formed between a serpin inhibitor and a serine proteinase.     

An interactive computer graphics study of thermolysin-catalyzed peptide cleavage and inhibition by N-carboxymethyl dipeptides

Interactive computer graphics was used as a tool in studying the cleavage mechanism of the model substrate Z-Phe-Phe-Leu-Trp by the zinc endopeptidase thermolysin. Two Michaelis complexes and three binding orientations of the tetrahedral intermediate to the crystal structure of thermolysin were investigated. Our results indicate that a Michaelis complex, which does not involve coordination of the scissile peptide to the zinc, is consistent with available experimental data and the most plausible of the two complexes. A tetrahedral intermediate complex wherein the two oxygens of the hydrated scissile peptide straddle the zinc in a bidentate fashion results in the most favorable interactions with the active site. The preferred tetrahedral intermediate and Michaelis complex provide a rationalization for the published substrate data. A trajectory for proceeding from the Michaelis complex to the tetrahedral intermediate is proposed. This trajectory involves a simultaneous activation of the zinc-bound water molecule concurrent with attack on the scissile peptide. A detailed ordered product release mechanism is also presented. These studies suggest some modifications and a number of extensions to the mechanism proposed earlier [Kester, W. R., & Matthews, B. W. (1977) Biochemistry 16, 2506; Holmes, M. A., & Matthews, B. W. (1981) Biochemistry 20, 6912]. The binding mode of the thermolysin inhibitor N-(1-carboxy-3-phenylpropyl)-L-leucyl-L-tryptophan [Monzingo, A. F., & Matthews, B. W. (1984) Biochemistry (preceding paper in this issue)] is compared with that of the preferred tetrahedral intermediate, providing insight into this inhibitor design.     

Stereochemical analysis of peptide bond hydrolysis catalyzed by the aspartic proteinase penicillopepsin

The X-ray crystal structures of native penicillopepsin and of its complex with a synthetic analogue of the inhibitor pepstatin have been refined recently at 1.8-A resolution. These highly refined structures permit a detailed examination of peptide hydrolysis in the aspartic proteinases. Complexes of penicillopepsin with substrate and catalytic intermediates were modeled, by using computer graphics, with minimal perturbation of the observed inhibitor complex. A thallium ion binding experiment shows that the position of solvent molecule O39, between Asp-33(32) and Asp-213(215) in the native structure, is favorable for cations, a fact that places constraints on possible mechanisms. A mechanism for hydrolysis is proposed in which Asp-213(215) acts as an electrophile by protonating the carbonyl oxygen of the substrate, thereby polarizing the carbon-oxygen bond, a water molecule bound to Asp-33(32) (O284 in the native structure) attacks the carbonyl carbon as the nucleophile in a general-base mechanism, the newly pyramidal peptide nitrogen is protonated, either from the solvent after nitrogen inversion or by an internal proton transfer via Asp-213(215) from a hydroxyl of the tetrahedral carbon, and the tetrahedral intermediate breaks down in a manner consistent with the stereoelectronic hypothesis. The models permit the rationalization of observed subsite preferences for substrates and may be useful in predicting subsite preferences of other aspartic proteinases.     

Extended substrate recognition in caspase-3 revealed by high resolution X-ray structure analysis

Caspases are cysteine proteases involved in the signalling cascades of programmed cell death in which caspase-3 plays a central role, since it propagates death signals from intrinsic and extrinsic stimuli to downstream targets. The atomic resolution (1.06 Angstroms) crystal structure of the caspase-3 DEVD-cmk complex reveals the structural basis for substrate selectivity in the S4 pocket. A low-barrier hydrogen bond is observed between the side-chains of the P4 inhibitor aspartic acid and Asp179 of the N-terminal tail of the symmetry related p12 subunit. Site-directed mutagenesis of Asp179 confirmed the significance of this residue in substrate recognition. In the 1.06 Angstroms crystal structure, a radiation damage induced rearrangement of the inhibitor methylketone moiety was observed. The carbon atom that in a substrate would represent the scissile peptide bond carbonyl carbon clearly shows a tetrahedral coordination and resembles the postulated tetrahedral intermediate of the acylation reaction.     

Caught in the Act: the 1.5 A resolution crystal structures of the HIV-1 protease and the I54V mutant reveal a tetrahedral reaction intermediate

HIV-1 protease (PR) is the target for several important antiviral drugs used in AIDS therapy. The drugs bind inside the active site cavity of PR where normally the viral polyprotein substrate is bound and hydrolyzed. We report two high-resolution crystal structures of wild-type PR (PRWT) and the multi-drug-resistant variant with the I54V mutation (PRI54V) in complex with a peptide at 1.46 and 1.50 A resolution, respectively. The peptide forms a gem-diol tetrahedral reaction intermediate (TI) in the crystal structures. Distinctive interactions are observed for the TI binding in the active site cavity of PRWT and PRI54V. The mutant PRI54V/TI complex has lost water-mediated hydrogen bond interactions with the amides of Ile50 and Ile50' in the flap. Hence, the structures provide insight into the mechanism of drug resistance arising from this mutation. The structures also illustrate an intermediate state in the hydrolysis reaction. One of the gem-diol hydroxide groups in the PRWT complex forms a very short (2.3 A) hydrogen bond with the outer carboxylate oxygen of Asp25. Quantum chemical calculations based on this TI structure are consistent with protonation of the inner carboxylate oxygen of Asp25', in contrast to several theoretical studies. These TI complexes and quantum calculations are discussed in relation to the chemical mechanism of the peptide bond hydrolysis catalyzed by PR.     

Molecular mechanics calculations on Rous sarcoma virus protease with peptide substrates.

Molecular models of Rous sarcoma virus (RSV) protease and 20 peptide substrates with single amino acid substitutions at positions from P4 to P3', where the scissile bond is between P1 and P1'. were built and compared with kinetic measurements. The unsubstituted peptide substrate. Pro-Ala-Val-Ser-Leu-Ala-Met-Thr, represents the NC-PR cleavage site of RSV protease. Models were built of two intermediates in the catalytic reaction, RSV protease with peptide substrate and with the tetrahedral intermediate. The energy minimization used an algorithm that increased the speed and eliminated a cutoff for nonbonded interactions. The calculated protease-substrate interaction energies showed correlation with the relative catalytic efficiency of peptide hydrolysis. The calculated interaction energies for the 8 RSV protease-substrate models with changes in P1 to P1' next to the scissile bond gave the highest correlation coefficient of 0.79 with the kinetic measurements, whereas all 20 substrates showed the lower, but still significant correlation of 0.46. Models of the tetrahedral reaction intermediates gave a correlation of 0.72 for the 8 substrates with changes next to the scissile bond, whereas a correlation coefficient of only 0.34 was observed for all 20 substrates. The differences between the energies calculated for the tetrahedral intermediate and the bound peptide gave the most significant correlation coefficients of 0.90 for models with changes in P1 and P1', and 0.56 for all substrates. These results are compared to those from similar calculations on HIV-1 protease and discussed in relation to the rate-limiting steps in the catalytic mechanism and the entropic contributions.     

Kinetic and crystallographic analysis of complexes formed between elastase and peptides from beta-casein.

Human beta-casomorphin-7 (NH2-Tyr-Pro-Phe-Val-Glu-Pro-Ile-CO2H) is a naturally occurring peptide inhibitor of elastase that has been shown to form an acyl-enzyme complex stable enough for X-ray crystallographic analysis at pH 5. To investigate the importance of the N-terminal residues of the beta-casomorphin-7 peptide for the inhibition of elastase, kinetic and crystallographic analyses were undertaken to identify the minimum number of residues required for effective formation of a stable complex between truncated beta-casomorphin-7 peptides and porcine pancreatic elastase (PPE). The results clearly demonstrate that significant inhibition of PPE can be effected by simple tri-, tetra-and pentapeptides terminating in a carboxylic acid. These results also suggest that in vivo regulation of protease activity could be mediated via short peptides as well as by proteins. Crystallographic analysis of the complex formed between N-acetyl-Val-Glu-Pro-Ile-CO2H and PPE at pH 5 (to 1.67 A resolution) revealed an active site water molecule in an analogous position to that observed in the PPE/beta-casomorphin-7 structure supportive of its assignment as the 'hydrolytic water' in the deacylation step of serine protease catalysis.     

Kinetics of the interaction of alpha-chymotrypsin with trypsin kallikrein inhibitor (Kunitz) in which the reactive-site peptide bond Lys-15--Ala-16 is split.

Modified trypsin kallikrein inhibitor (I*), with the reactive-site peptide bond Lys-15--Ala-16 split, reacts with alpha-chymotrypsin (E) via an intermediate X to the stable tetrahedral complex C:E + I in equilibrium X leads to C. Formation X constitutes a fast pre-equilibrium (equilibrium constant Kx = 7 X 10(-5) M, association rate constant kx = 4 X 10(3)M-1s-1) to the slow reaction X leads to C (rate constant kc = 2 X 10(-3) s-1), all values at pH 7.5. No intermediate X is observed when alpha-chymotrypsin reacts with I*-OMe in which the carboxyl group of Lys-15 is esterified by methanol. This observation as well as the different pH dependence of the overall association rate constants in the case of I* and I*-OMe indicate tha formation of X precedes formation of the acyl enzyme in the catalytic pathway. The data are compared to the similar results obtained with beta-trypsin and I* or I*-OMe.     

The catalytic mechanism of aspartic proteinases

The highly symmetric active site of an aspartic proteinase, endothiapepsin, binds a water molecule ideally situated for nucleophilic attack on a substrate peptide bond whose distortion from planarity is stabilised by interactions of the substrate with the extended binding cleft. The apparent electrophilicity of the catalysis results from this distortion. The scissile peptide bond is orientated with the carbonyl oxygen hydrogen bonding to the tip of the beta-hairpin 'flap' which lies over the cleft. Nucleophilic attack by the bound water leads to a tetrahedral intermediate similar to observed complexes with hydroxyl inhibitors and stabilised by hydrogen bonds with the flap.     

Theoretical study on isomerization and peptide bond cleavage at aspartic residue

Isomerization and peptide bond cleavage at aspartic residue (Asp) in peptide models have been reported. In this study, the mechanisms and energies concerning the isomerization and peptide bond cleavage at Asp residue were investigated by the density functional theory (DFT) at B3LYP/6-311++G(d,p). The integral equation formalism-polarizable continuum model (IEF-PCM) was utilized to calculate solvation effect by single-point calculation of the gas-phase B3LYP/6-311++G(d,p)-optimized structure. Mechanisms and energies of the dehydration in isomerization reaction of Asp residue were comparatively analyzed with the deamidation reaction of Asn residue. The results show that the succinimide intermediate was formed preferentially through the step-wise reaction via the tetrahedral intermediate. The cleavage at C-terminus is more preferential than those at N-terminus. In comparison to isomerization, peptide bond cleavage is ～20 kcal mol^?1 and lower in activation barrier than the isomerization. So, in this case, the isomerization of Asp is inhibited by the peptide bond cleavage.     

Kinetic isotope effect analysis of the ribosomal peptidyl transferase reaction

The ribosome is the macromolecular machine responsible for protein synthesis in all cells. Here, we establish a kinetic framework for the 50S modified fragment reaction that makes it possible to measure the kinetic effects that result from isotopic substitution in either the A or P site of the ribosome. This simplified peptidyl transferase assay follows a rapid equilibrium random mechanism in which the reverse reaction is nonexistent and the forward commitment is negligible. A normal effect (1.009) is observed for (15)N substitution of the incoming nucleophile at both low and high pH. This suggests that the first irreversible step is the formation of the tetrahedral intermediate. The observation of a normal isotope effect that does not change as a function of pH suggests that the ribosome promotes peptide bond formation by a mechanism that differs in its details from an uncatalyzed aminolysis reaction in solution. This implies that the ribosome contributes chemically to catalysis of peptide bond formation.     

The 1.8 A structure of the complex between chymostatin and Streptomyces griseus protease A. A model for serine protease catalytic tetrahedral intermediates

The naturally occurring serine protease inhibitor, chymostatin, forms a hemiacetal adduct with the catalytic Ser195 residue of Streptomyces griseus protease A. Restrained parameter least-squares refinement of this complex to 1.8 A resolution has produced an R index of 0 X 123 for the 11,755 observed reflections. The refined distance of the carbonyl carbon atom of the aldehyde to O gamma of Ser195 is 1 X 62 A. Both the R and S configurations of the hemiacetal occur in equal populations, with the end result resembling the expected configuration for a covalent tetrahedral product intermediate of a true substrate. This study strengthens the concept that serine proteases stabilize a covalent, tetrahedrally co-ordinated species and elaborates those features of the enzyme responsible for this effect. We propose that a major driving force for the hydrolysis of peptide bonds by serine proteases is the non-planar distortion of the scissile bond by the enzyme, which thereby lowers the activation energy barrier to hydrolysis by eliminating the resonance stabilization energy of the peptide bond.     

Mechanism-based inactivation of human leukocyte elastase via an enzyme-induced sulfonamide fragmentation process

We describe herein the design and in vitro biochemical evaluation of a novel class of mechanism-based inhibitors of human leukocyte elastase (HLE) that inactivate the enzyme via an unprecedented enzyme-induced sulfonamide fragmentation cascade. The inhibitors incorporate in their structure an appropriately functionalized saccharin scaffold. Furthermore, the inactivation of the enzyme by these inhibitors was found to be time-dependent and to involve the active site. Biochemical, HPLC, and mass spectrometric studies show that the interaction of these inhibitors with HLE results in the formation of a stable acyl complex and is accompanied by the release of (L) phenylalanine methyl ester. The data are consistent with initial formation of a Michaelis-Menten complex and subsequent formation of a tetrahedral intermediate with the active site serine (Ser(195)). Collapse of the tetrahedral intermediate with tandem fragmentation results in the formation of a highly reactive conjugated sulfonyl imine which can either react with water to form a stable acyl enzyme and/or undergo a Michael addition reaction with an active site nucleophilic residue (His(57)). It is also demonstrated herein that this class of compounds can be used in the design of inhibitors of serine proteases having either a neutral or basic primary substrate specificity. Thus, the results suggest that these inhibitors constitute a potential general class of mechanism-based inhibitors of (chymo)trypsin-like serine proteases.     

Dipeptide hydrolysis by the dinuclear zinc enzyme human renal dipeptidase: Mechanistic insights from DFT calculations

The reaction mechanism of the dinuclear zinc enzyme human renal dipeptidase is investigated using hybrid density functional theory. This enzyme catalyzes the hydrolysis of dipeptides and β-lactam antibiotics. Two different protonation states in which the important active site residue Asp288 is either neutral or ionized were considered. In both cases, the bridging hydroxide is shown to be capable of performing the nucleophilic attack on the substrate carbonyl carbon from its bridging position, resulting in the formation of a tetrahedral intermediate. This step is followed by protonation of the dipeptide nitrogen, coupled with C-N bond cleavage. The calculations establish that both cases have quite feasible energy barriers. When the Asp288 is neutral, the hydrolytic reaction occurs with a large exothermicity. However, the reaction becomes very close to thermoneutral with an ionized Asp288. The two zinc ions are shown to play different roles in the reaction. Zn1 binds the amino group of the substrate, and Zn2 interacts with the carboxylate group of the substrate, helping in orienting it for the nucleophilic attack. In addition, Zn2 stabilizes the oxyanion of the tetrahedral intermediate, thereby facilitating the nucleophilic attack.