A theoretical study of the active sites of papain and S195C rat trypsin: implications for the low reactivity of mutant serine proteinases.

The serine and cysteine proteinases represent two important classes of enzymes that use a catalytic triad to hydrolyze peptides and esters. The active site of the serine proteinases consists of three key residues, Asp...His...Ser. The hydroxyl group of serine functions as a nucleophile and the imidazole ring of histidine functions as a general acid/general base during catalysis. Similarly, the active site of the cysteine proteinases also involves three key residues: Asn, His, and Cys. The active site of the cysteine proteinases is generally believed to exist as a zwitterion (Asn...His+...Cys-) with the thiolate anion of the cysteine functioning as a nucleophile during the initial stages of catalysis. Curiously, the mutant serine proteinases, thiol subtilisin and thiol trypsin, which have the hybrid Asp...His...Cys triad, are almost catalytically inert. In this study, ab initio Hartree-Fock calculations have been performed on the active sites of papain and the mutant serine proteinase S195C rat trypsin. These calculations predict that the active site of papain exists predominately as a zwitterion (Cys-...His+...Asn). However, similar calculations on S195C rat trypsin demonstrate that the thiol mutant is unable to form a reactive thiolate anion prior to catalysis. Furthermore, structural comparisons between native papain and S195C rat trypsin have demonstrated that the spatial juxtapositions of the triad residues have been inverted in the serine and cysteine proteinases and, on this basis, I argue that it is impossible to convert a serine proteinase to a cysteine proteinase by site-directed mutagenesis.     

The role of the conserved box E residues in the active site of the Escherichia coli type I signal peptidase

Type I signal peptidases are integral membrane proteins that function to remove signal peptides from secreted and membrane proteins. These enzymes carry out catalysis using a serine/lysine dyad instead of the prototypical serine/histidine/aspartic acid triad found in most serine proteases. Site-directed scanning mutagenesis was used to obtain a qualitative assessment of which residues in the fifth conserved region, Box E, of the Escherichia coli signal peptidase I are critical for maintaining a functional enzyme. First, we find that there is no requirement for activity for a salt bridge between the invariant Asp-273 and the Arg-146 residues. In addition, we show that the conserved Ser-278 is required for optimal activity as well as conserved salt bridge partners Asp-280 and Arg-282. Finally, Gly-272 is essential for signal peptidase I activity, consistent with it being located within van der Waals proximity to Ser-278 and general base Lys-145 side-chain atoms. We propose that replacement of the hydrogen side chain of Gly-272 with a methyl group results in steric crowding, perturbation of the active site conformation, and specifically, disruption of the Ser-90/Lys-145 hydrogen bond. A refined model is proposed for the catalytic dyad mechanism of signal peptidase I in which the general base Lys-145 is positioned by Ser-278, which in turn is held in place by Asp-280.     

Interactions between Asp, His, Ser residues within models of the active site of serine proteases. A theoretical empirical study

Empirical theoretical calculations have been performed on a simplified model of the active site of two serine proteases: alpha-chymotrypsin and subtilisin Novo. The stability of the catalytic triad and the hydrogen bond formation between the Asp-His and His-Ser pairs have been examined for different protonation states. The results show that the Asp-His interactions prevail upon the His-Ser ones. Agreement between calculated configurations and the crystal structure of the site suggests that the presence of other residues near the functional residues is not determinant for the stability of the triad in alpha-chymotrypsin. In subtilisin Novo, on the contrary, the presence of the neighbouring residues seems to contribute more largely to the stability. Strong hydrogen bond interactions between the His and Ser residues do not exist in the resting enzymes. Any improvement of the His-Ser interactions requires large destabilization of the Asp-His diad. Our results suggest that the mechanism of the proton transfer can occur only from perturbations of the active site structure induced by the presence of the substrate.     

Structure of proline iminopeptidase from Xanthomonas campestris pv. citri: a prototype for the prolyl oligopeptidase family.

The proline iminopeptidase from Xanthomonas campestris pv. citri is a serine peptidase that catalyses the removal of N-terminal proline residues from peptides with high specificity. We have solved its three-dimensional structure by multiple isomorphous replacement and refined it to a crystallographic R-factor of 19.2% using X-ray data to 2.7 A resolution. The protein is folded into two contiguous domains. The larger domain shows the general topology of the alpha/beta hydrolase fold, with a central eight-stranded beta-sheet flanked by two helices and the 11 N-terminal residues on one side, and by four helices on the other side. The smaller domain is placed on top of the larger domain and essentially consists of six helices. The active site, located at the end of a deep pocket at the interface between both domains, includes a catalytic triad of Ser110, Asp266 and His294. Cys269, located at the bottom of the active site very close to the catalytic triad, presumably accounts for the inhibition by thiol-specific reagents. The overall topology of this iminopeptidase is very similar to that of yeast serine carboxypeptidase. The striking secondary structure similarity to human lymphocytic prolyl oligopeptidase and dipeptidyl peptidase IV makes this proline iminopeptidase structure a suitable model for the three-dimensional structure of other peptidases of this family.     

Structure of wheat serine carboxypeptidase II at 3.5-A resolution. A new class of serine proteinase

The structure of serine carboxypeptidase II from wheat bran has been determined to 3.5-A resolution by multiple isomorphous replacement, solvent flattening, and crystallographic refinement. The amino acid sequence has been fit to the electron density map and the model refined to a conventional crystallographic R factor of 20.9%. The molecule is an alpha + beta protein and contains a "catalytic triad" (Asp338, His397, and Ser146) similar in arrangement to those in chymotrypsin and subtilisin. The -fold of the polypeptide backbone is, however, completely different from those enzymes. This suggests that this is a third example of convergent evolution to a common enzymatic mechanism. The -fold is, on the other hand, surprisingly similar to that of the zinc proteinase carboxypeptidase A.     

A catalytic diad involved in substrate-assisted catalysis: NMR study of hydrogen bonding and dynamics at the active site of phosphatidylinositol-specific phospholipase C

Phosphatidylinositol-specific phospholipase Cs (PI-PLCs, EC 3.1.4.10) are ubiquitous enzymes that cleave phosphatidylinositol or phosphorylated derivatives, generating second messengers in eukaryotic cells. A catalytic diad at the active site of Bacillus cereus PI-PLC composed of aspartate-274 and histidine-32 was postulated from the crystal structure to form a catalytic triad with the 2-OH group of the substrate [Heinz, D. W., et al. (1995) EMBO J. 14, 3855-3863]. This catalytic diad has been observed directly by proton NMR. The single low-field line in the 1H NMR spectrum is assigned by site-directed mutagenesis: The peak is present in the wild type but absent in the mutants H32A and D274A, and arises from the histidine Hdelta1 forming the Asp274-His32 hydrogen bond. This hydrogen is solvent-accessible, and exchanges slowly with H2O on the NMR time scale. The position of the low-field peak shifts from 16.3 to 13.8 ppm as the pH is varied from 4 to 9, reflecting a pKa of 8.0 at 6 degrees C, which is identified with the pKa of His32. The Hdelta1 signal is modulated by rapid exchange of the Hepsilon2 with the solvent. Estimates of the exchange rate as a function of pH and protection factors are derived from a line shape analysis. The NMR behavior is remarkably similar to that of the serine proteases. The postulated function of the Asp274-His32 diad is to hydrogen-bond with the 2-OH of phosphatidylinositol (PI) substrate to form a catalytic triad analogous to Asp-His-Ser of serine proteases. This is an example of substrate-assisted catalysis where the substrate provides the catalytic nucleophile of the triad. This hydrogen bond becomes shorter as the imidazole is protonated, suggesting it is stronger in the transition state, contributing further to the catalytic efficiency. The hydrogen bond fits the NMR criteria for a short, strong hydrogen bond, i.e., a highly deshielded proton resonance, bond length of 2.64 +/- 0.04 A at pH 6 measured by NMR, a D/H fractionation factor significantly lower than 1.0, and a protection factor > or = 100.     

The noncatalytic triad of alpha-amylases: a novel structural motif involved in conformational stability

Chloride-activated alpha-amylases contain a noncatalytic triad, independent of the glycosidic active site, perfectly mimicking the catalytic triad of serine-proteases and of other active serine hydrolytic enzymes. Mutagenesis of Glu, His, and Ser residues in various alpha-amylases shows that this pattern is a structural determinant of the enzyme conformation that cannot be altered without losing the intrinsic stability of the protein. (1)H-(15)N NMR spectra of a bacterial alpha-amylase reveal proton signals that are identical with the NMR signature of catalytic triads and especially a deshielded proton involving a protonated histidine and displaying properties similar to that of a low barrier hydrogen bond. It is proposed that the H-bond between His and Glu of the noncatalytic triad is an unusually strong interaction, responsible for the observed NMR signal and for the weak stability of the triad mutants. Furthermore, a stringent template-based search of the Protein Data Bank demonstrated that this motif is not restricted to alpha-amylases, but is also found in 80 structures from 33 different proteins, amongst which SH2 domain-containing proteins are the best representatives.     

Unconventional serine proteases: variations on the catalytic Ser/His/Asp triad configuration

Serine proteases comprise nearly one-third of all known proteases identified to date and play crucial roles in a wide variety of cellular as well as extracellular functions, including the process of blood clotting, protein digestion, cell signaling, inflammation, and protein processing. Their hallmark is that they contain the so-called "classical" catalytic Ser/His/Asp triad. Although the classical serine proteases are the most widespread in nature, there exist a variety of "nonclassical" serine proteases where variations to the catalytic triad are observed. Such variations include the triads Ser/His/Glu, Ser/His/His, and Ser/Glu/Asp, and include the dyads Ser/Lys and Ser/His. Other variations are seen with certain serine and threonine peptidases of the Ntn hydrolase superfamily that carry out catalysis with a single active site residue. This work discusses the structure and function of these novel serine proteases and threonine proteases and how their catalytic machinery differs from the prototypic serine protease class.     

The unusual catalytic triad of poliovirus protease 3C

Picornaviruses are small pathogen RNA viruses, like poliovirus, hepatitis A virus, rhinovirus, and others. They produce a large polyprotein, which is cleaved by virally encoded cysteine peptidases, picornains 2A and 3C. Picornain 3C represents an intermediate between the serine peptidase chymotrypsin and the cysteine peptidase papain. Its steric structure resembles chymotrypsin, but its nucleophile is a thiol instead of the hydroxyl group. The histidine is a general base catalyst in chymotrypsin but forms a thiolate-imidazolium ion pair in papain. The third member of the catalytic triad is an acid (Glu71) as in chymotrypsin rather than an amide found in papain. Transformation of poliovirus 3C peptidase into a serine peptidase results in lower activity by a factor of 430, but the activity extends toward higher pH with the more basic hydroxyl group. The decrease in activity is caused by the less ordered active site, as supported by the unfavorable entropy of activation. At 25 degrees C the specificity rate constant for the thiol enzyme approaches k(1), the rate constant for the formation of the enzyme-substrate complex, but k(2), the acylation constant, becomes predominant with the increase in temperature. In contrast, for the serine peptidase the specificity constant is less than k(1) over the entire temperature range, and the transition state is controlled by both k(1) and k(2). The acidic component of the catalytic triad is essential for activity, but its negative charge does not influence the ionization of the thiol group.     

Molecular cloning and sequence analysis of human dipeptidyl peptidase IV, a serine proteinase on the cell surface.

The cDNA coding for the human dipeptidyl peptidase IV (DPPIV) has been isolated and sequenced. The nucleotide sequence (3465 bp) of the cDNA contains an open reading frame encoding a polypeptide comprising 766 amino acids, one residue less than those of rat DPPIV. The predicted amino acid sequence exhibits 84.9% identity to that of the rat enzyme, and contains nine potential N-linked glycosylation sites, one site more than those in the rat enzyme. A putative catalytic triad for serine proteinases, serine, aspartic acid and histidine, are found in a completely conserved COOH-terminal region (positions 625-752).     

Saturation mutagenesis reveals that GLU54 of norovirus 3C-like protease is not essential for the proteolytic activity

The norovirus 3C-like protease is a member of the chymotrypsin-like serine protease superfamily. Previous characterization of its crystal structure has implicated the Glu54-His30-Cys139 triad in the catalysis. In the present study, the Glu54 residue of the protease was subjected to site-saturation mutagenesis, with the result that nearly half of the mutants retained the significant proteolytic activity. It was suggested that a carboxylate at position 54 was not essential for the activity. The in vitro assays of the proteolysis revealed that most of Glu54 mutants retained relatively high proteolytic activity. When the Glu54 mutation was combined with the Ser mutation of the Cys139 residue, a nucleophile, only the Asp54 and Gln54 mutations showed proteolytic activity comparable to that of the Ser139 single mutant, suggesting that a hydrogen bond between Glu54 and His30 was critical in the Ser139 background. These results suggested that the mechanism of the proteolysis by the wild-type norovirus 3C-like protease was different from that of typical chymotrypsin-like serine proteases.     

Structural changes of phenylalanine 338 and histidine 447 revealed by the crystal structures of tabun-inhibited murine acetylcholinesterase

Organophosphorus compounds (OPs) interfere with the catalytic mechanism of acetylcholinesterase (AChE) by rapidly phosphorylating the catalytic serine residue. The inhibited enzyme can at least partly be reactivated with nucleophilic reactivators such as oximes. The covalently attached OP conjugate may undergo further intramolecular dealkylation or deamidation reactions, a process termed "aging" that results in an enzyme considered completely resistant to reactivation. Of particular interest is the inhibition and aging reaction of the OP compound tabun since tabun conjugates display an extraordinary resistance toward most reactivators of today. To investigate the structural basis for this resistance, we determined the crystal structures of Mus musculus AChE (mAChE) inhibited by tabun prior to and after the aging reaction. The nonaged tabun conjugate induces a structural change of the side chain of His447 that uncouples the catalytic triad and positions the imidazole ring of His447 in a conformation where it may form a hydrogen bond to a water molecule. Moreover, an unexpected displacement of the side chain of Phe338 narrows the active site gorge. In the crystal structure of the aged tabun conjugate, the side chains of His447 and Phe338 are reversed to the conformation found in the apo structure of mAChE. A hydrogen bond between the imidazole ring of His447 and the ethoxy oxygen of the aged tabun conjugate stabilizes the side chain of His447. The displacement of the side chain of Phe338 into the active site gorge of the nonaged tabun conjugate may interfere with the accessibility of reactivators and thereby contribute to the high resistance of tabun conjugates toward reactivation.     

Structure determinants and substrate recognition of serine carboxypeptidase-like acyltransferases from plant secondary metabolism

Structures of the serine carboxypeptidase-like enzymes 1-O-sinapoyl-beta-glucose:L-malate sinapoyltransferase (SMT) and 1-O-sinapoyl-beta-glucose:choline sinapoyltransferase (SCT) were modeled to gain insight into determinants of specificity and substrate recognition. The structures reveal the alpha/beta-hydrolase fold as scaffold for the catalytic triad Ser-His-Asp. The recombinant mutants of SMT Ser173Ala and His411Ala were inactive, whereas Asp358Ala displayed residual activity of 20%. 1-O-sinapoyl-beta-glucose recognition is mediated by a network of hydrogen bonds. The glucose moiety is recognized by a hydrogen bond network including Trp71, Asn73, Glu87 and Asp172. The conserved Asp172 at the sequence position preceding the catalytic serine meets sterical requirements for the glucose moiety. The mutant Asn73Ala with a residual activity of 13% underscores the importance of the intact hydrogen bond network. Arg322 is of key importance by hydrogen bonding of 1-O-sinapoyl-beta-glucose and L-malate. By conformational change, Arg322 transfers L-malate to a position favoring its activation by His411. Accordingly, the mutant Arg322Glu showed 1% residual activity. Glu215 and Arg219 establish hydrogen bonds with the sinapoyl moiety. The backbone amide hydrogens of Gly75 and Tyr174 were shown to form the oxyanion hole, stabilizing the transition state. SCT reveals also the catalytic triad and a hydrogen bond network for 1-O-sinapoyl-beta-glucose recognition, but Glu274, Glu447, Thr445 and Cys281 are crucial for positioning of choline.     

Carboxyl proteinase from Pseudomonas defines a novel family of subtilisin-like enzymes

The crystal structure of a pepstatin-insensitive carboxyl proteinase from Pseudomonas sp. 101 (PSCP) has been solved by single-wavelength anomalous diffraction using the absorption peak of bromide anions. Structures of the uninhibited enzyme and of complexes with an inhibitor that was either covalently or noncovalently bound were refined at 1.0-1.4 A resolution. The structure of PSCP comprises a single compact domain with a diameter of approximately 55 A, consisting of a seven-stranded parallel beta-sheet flanked on both sides by a number of helices. The fold of PSCP is a superset of the subtilisin fold, and the covalently bound inhibitor is linked to the enzyme through a serine residue. Thus, the structure of PSCP defines a novel family of serine-carboxyl proteinases (defined as MEROPS S53) with a unique catalytic triad consisting of Glu 80, Asp 84 and Ser 287.     

Prediction of the secondary structures of stefins and cystatins, the low-molecular mass protein inhibitors of cysteine proteinases

A procedure for classifying proteins of known sequence into structurally similar groups was developed on the basis of the Argos parametric approach. It is shown that stefins and cystatins constitute two structurally well resolved, but homologous groups of proteins. Furthermore, it is very probable that segments of secondary structures within each family are conserved, although significant differences between stefins and cystatins are indicated at the level of secondary structure. Next, secondary structures of all sequenced stefins and cystatins were predicted and used in the construction of secondary structures of the "typical stefin" and the "typical cystatin". Results were interpreted in the light of evolution and inhibition mechanism: Alignment of the "typical stefin" versus the "typical cystatin" secondary structure segments suggests that the divergence of stefin and cystatin families did not occur by a gene fusion event, but only by a mechanism of substitution, insertion and/or deletion. The central region of low-molecular mass cystatins, which is assumed to interact with cysteine proteinases, is predicted to be in a beta-sheet conformation. This resembles the beta-sheet in the active site of "standard mechanism" serine proteinases inhibitors.     

The Bacillus subtilis Signaling Protein SpoIVB Defines a New Family of Serine Peptidases

The protein SpoIVB plays a key role in signaling in the final sigma(K) checkpoint of Bacillus subtilis. This regulatory mechanism coordinates late gene expression during development in this organism and we have recently shown SpoIVB to be a serine peptidase. SpoIVB signals by transiting a membrane, undergoing self-cleavage, and then by an unknown mechanism activating a zinc metalloprotease, SpoIVFB, which cleaves pro-final sigma(K) to its active form, final sigma(K), in the outer mother cell chamber of the developing cell. In this work we have characterized the serine peptidase domain of SpoIVB. Alignment of SpoIVB with homologues from other spore formers has allowed site-specific mutagenesis of all potential active site residues within the peptidase domain. We have defined the putative catalytic domain of the SpoIVB serine peptidase as a 160-amino-acid residue segment at the carboxyl terminus of the protein. His236 and Ser378 are the most important residues for proteolysis, with Asp363 being the most probable third member of the catalytic triad. In addition, we have shown that mutations at residues Asn290 and His394 lead to delayed signaling in the final sigma(K) checkpoint. The active site residues suggest that SpoIVB and its homologues from other spore formers are members of a new family of serine peptidases of the trypsin superfamily.     

Mapping of the epitope of a monoclonal antibody protecting plasminogen activator inhibitor-1 against inactivating agents.

Plasminogen activator inhibitor-1 (PAI-1) belongs to the serpin family of serine proteinase inhibitors. Serpins inhibit their target proteinases by an ester bond being formed between the active site serine of the proteinase and the P1 residue of the reactive centre loop (RCL) of the serpin, followed by insertion of the RCL into beta-sheet A of the serpin. Concomitantly, there are conformational changes in the flexible joint region lateral to beta-sheet A. We have now, by site-directed mutagenesis, mapped the epitope for a monoclonal antibody, which protects the inhibitory activity of PAI-1 against inactivation by a variety of agents acting on beta-sheet A and the flexible joint region. Curiously, the epitope is localized in alpha-helix C and the loop connecting alpha-helix I and beta-strand 5A, on the side of PAI-1 opposite to beta-sheet A and distantly from the flexible joint region. By a combination of site-directed mutagenesis and antibody protection against an inactivating organochemical ligand, we were able to identify a residue involved in conferring the antibody-induced conformational change from the epitope to the rest of the molecule. We have thus provided evidence for communication between secondary structural elements not previously known to interact in serpins.     

The basic difference in catalyses by serine and cysteine proteinases resides in charge stabilization in the transition state

Besides the mechanistic similarities, in particular acylenzyme formation, kinetic investigations and X-ray diffraction studies have revealed some differences between the mechanisms of serine and cysteine proteinases: general base-catalysis in acylation, catalytic contribution by oxyanion binding, and a negatively charged catalytic triad in serine proteinases, but not in cysteine proteinases. In this paper we point out that all these differences are related and connected with the mode of stabilization of the zwitterionic species developing in the transition state of the reactions. In the case of serine proteinases this charge separation requires facilitation by the oxyanion binding and the negative charge of the catalytic triad. On the other hand cysteine proteinases do not require such contributions as they are capable of stabilizing the ion-pair even in the ground state of the reaction. Therefore, cysteine proteinases, in contrast to serine proteinases, may be regarded as "activated" enzymes.     

Isolation and properties of a metal-dependent endopeptidase from human uterus hydrolysing synthetic collagenase substrates.

A metal-dependent peptidase was isolated from the homogenate of human uterus by standard chromatographic techniques and purified to apparent homogeneity. The peptidase hydrolysed the synthetic vertebrate collagenase substrate 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg (Dnp-peptide), the synthetic bacterial collagenase substrate 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg (PZ-peptide) and gelatinolytic peptides of gelatin, but was inactive against collagen type I, gelatin and casein. The cleavage site for the Dnp-peptide was the Gly-Ile bond. The enzyme was not only inhibited by metal chelators, such as EDTA, 1,10-phenantroline and dithiothreitol but also by thiol reagents, such as mersalylic acid and N-ethylmaleimid. However, E-64, an inhibitor for thiolproteinases, and leupeptin, an inhibitor for thiol- and serine proteases, did not exhibit any inhibitory activity. Pepstatin, an inhibitor for aspartate proteinases, and inhibitors for serine proteinases like phenylmethanesulfonyl fluoride and Trasylol were ineffective as well. The purified peptidase displayed a single band in the SDS-PAGE with an apparent molecular mass of 65 kDa. Employing isoelectric focusing an IP of 5.0 could be determined. The enzyme's properties are discussed in relation to the proteinase EC 3.4.24.11 and to proteinases of the collagenase family as well as the possibility to discriminate these three metalloproteinase classes by employing the Dnp-peptide.     

Visualization of the phosphorylated active site loop of the cytoplasmic B domain of the mannitol transporter II(Mannitol) of the Escherichia coli phosphotransferase system by NMR spectroscopy and residual dipolar couplings

The solution structure of a stably phosphorylated form of the cytoplasmic B domain of the mannitol-specific transporter (IIB(Mtl)) of the Escherichia coli phosphotransferase system, containing a mutation of the active site Cys384 to Ser, has been solved by NMR. The strategy employed relies principally on backbone residual dipolar couplings recorded in three different alignment media, supplemented by nuclear Overhauser enhancement data and torsion angle restraints related specifically to the active site loop (residues 383-393). As judged from the dipolar coupling data, the remainder of the structure is unchanged upon phosphorylation within the errors of the coordinates of the previously determined solution structure of unphosphorylated wild-type IIB(Mtl). Thus, only the active site loop was refined. Phosphorylation results in a backbone atomic rms shift of approximately 0.7 angstroms in the active site loop. The resulting conformation is less than 0.5 angstroms away from the equivalent P-loop in both the low and high molecular mass eukaryotic tyrosine phosphatases. 3J(NP) coupling constant measurements using quantitative J-correlation spectroscopy provide a direct demonstration of a hydrogen bond between the phosphoryl group and the backbone amide of Ser391 at position i + 7 from phospho-Ser384, with an approximately linear P-O-H(N) bond angle. The structure also reveals additional hydrogen bonding interactions involving the backbone amides of residues at positions i + 4 and i + 5, and the hydroxyl groups of two serine residues at positions i + 6 and i + 7 that stabilize the phosphoryl group.