Subsite preferences of pepstatin-insensitive carboxyl proteinases from prokaryotes: kumamolysin, a thermostable pepstatin-insensitive carboxyl proteinase

Kumamolysin, a carboxyl proteinase from Bacillus novosp. MN-32, is characterized by its thermostability and insensitivity to aspartic proteinase inhibitors such as pepstatin, diazoacetyl-DL-norleucine methylester, and 1,2-epoxy-3-(p-nitro-phenoxy)propane. Here, its substrate specificity was elucidated using two series of synthetic chromogenic substrates: P(5)-P(4)-P(3)-P(2)-Phe*Nph (p-nitrophenylalanine: *cleavage site)-P(2)'-P(3)', in which the amino acid residues at the P(5)-P(2), P(2)' and P(3)' positions were systematically substituted. Among 74 substrates, kumamolysin was shown to hydrolyze Lys-Pro-Ile-Pro-Phe-Nph-Arg-Leu most effectively. The kinetic parameters of this peptide were K(m) = 41+/-5 microM, k(cat) = 176+/- 10 s(-1), and k(cat)/K(m) = 4.3+/-0.6 mM(-1) x s(-1). These systematic analyses revealed the following features: (i) Kumamolysin had a unique preference for the P(2) position. Kumamolysin preferentially hydrolyzed peptides having an Ala or Pro residue at the P(2) position; this was also observed for the pepstatin-insensitive carboxyl proteinase from Bacillus coagulans J-4 [J-4; Shibata et al. (1998) J. Biochem. 124, 642-647]. Other carboxyl proteinases, including Pseudomonas sp. 101 pepstatin-insensitive carboxyl proteinase (PCP) and Xanthomonas sp. T-22 pepstatin-insensitive carboxyl proteinase (XCP), preferred peptides having hydrophobic and bulky amino acid residue such as Leu at the P(2) position. (ii) Kumamolysin preferred such charged amino acid residues as Glu or Arg at the P(2)' position, suggesting that the S(2)' subsite of kumamolysin is occupied by hydrophilic residues, similar to that of PCP, XCP, and J-4. In general, the S(2)' subsite of pepstatin-sensitive carboxyl proteinases (aspartic proteinases) is hydrophobic in nature. Thus, the hydrophilic nature of the S(2)' subsite was confirmed to be a distinguishing feature of pepstatin-insensitive carboxyl proteinases from prokaryotes.     

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.     

The 1.4 a crystal structure of kumamolysin: a thermostable serine-carboxyl-type proteinase

Kumamolysin is a thermostable endopeptidase from Bacillus novosp. MN-32, exhibiting maximal proteolytic activity around pH 3. It belongs to the newly identified family of serine-carboxyl proteinases, which also includes CLN2, a human lysosomal homolog recently implicated in a fatal neurodegenerative disease. Kumamolysin and its complexes with two aldehyde inhibitors were crystallized, and their three-dimensional structures were solved and refined with X-ray data to 1.4 A resolution. As its Pseudomonas homolog, kumamolysin exhibits a Ser/Glu/Asp catalytic triad with particularly short interconnecting hydrogen bonds and an oxyanion hole enabling the reactive serine to attack substrate peptide bonds at quite acidic pH. An additional Glu/Trp pair, unique to kumamolysin, might further facilitate proton delocalization during nucleophilic attack, in particular at high temperature.     

Processing, catalytic activity and crystal structures of kumamolisin-As with an engineered active site

Kumamolisin-As is an acid collagenase with a subtilisin-like fold. Its active site contains a unique catalytic triad, Ser278-Glu78-Asp82, and a putative transition-state stabilizing residue, Asp164. In this study, the mutants D164N and E78H/D164N were engineered in order to replace parts of the catalytic machinery of kumamolisin-As with the residues found in the equivalent positions in subtilisin. Unlike the wild-type and D164N proenzymes, which undergo instantaneous processing to produce their 37-kDa mature forms, the expressed E78H/D164N proenzyme exists as an equilibrated mixture of the nicked and intact forms of the precursor. X-ray crystallographic structures of the mature forms of the two mutants showed that, in each of them, the catalytic Ser278 makes direct hydrogen bonds with the side chain of Asn164. In addition, His78 of the double mutant is distant from Ser278 and Asp82, and the catalytic triad no longer exists. Consistent with these structural alterations around the active site, these mutants showed only low catalytic activity (relative k(cat) at pH 4.0 1.3% for D164N and 0.0001% for E78H/D164N). pH-dependent kinetic studies showed that the single D164N substitution did not significantly alter the logk(cat) vs. pH and log(k(cat)/Km) vs. pH profiles of the enzyme. In contrast, the double mutation resulted in a dramatic switch of the logk(cat) vs. pH profile to one that was consistent with catalysis by means of the Ser278-His78 dyad and Asn164, which may also account for the observed ligation/cleavage equilibrium of the precursor of E78H/D164N. These results corroborate the mechanistic importance of the glutamate-mediated catalytic triad and oxyanion-stabilizing aspartic acid residue for low-pH peptidase activity of the enzyme.     

Catalytic role of proton transfers in the formation of a tetrahedral adduct in a serine carboxyl peptidase

Quantum mechanical/molecular mechanical molecular dynamics and 2D free energy simulations are performed to study the formation of a tetrahedral adduct by an inhibitor N-acetyl-isoleucyl-prolyl-phenylalaninal (AcIPF) in a serine-carboxyl peptidase (kumamolisin-As) and elucidate the role of proton transfers during the nucleophilic attack by the Ser278 catalytic residue. It is shown that although the serine-carboxyl peptidases have a fold resembling that of subtilisin, the proton transfer processes during the nucleophilic attack by the Ser residue are likely to be more complex for these enzymes compared to the case in classical serine proteases. The computer simulations demonstrate that both general base and acid catalysts are required for the formation and stabilization of the tetrahedral adduct. The 2D free energy maps further demonstrate that the proton transfer from Ser278 to Glu78 (the general base catalyst) is synchronous with the nucleophilic attack, whereas the proton transfer from Asp164 (the general acid catalyst) to the inhibitor is not. The dynamics of the protons at the active site in different stages of the nucleophilic attack as well as the motions of the corresponding functional groups are also studied. It is found that the side chain of Glu78 is generally rather flexible, consistent with its possible multifunctional role during catalysis. The effects of proton shuffling from Asp82 to Glu78 and from Glu32 to Asp82 are examined, and the results indicate that such proton shuffling may not play an important role in the stabilization of the tetrahedral intermediate analogue.     

Crystallographic and biochemical investigations of kumamolisin-As, a serine-carboxyl peptidase with collagenase activity

Kumamolisin-As (previously called ScpA) is the first known example of a collagenase from the sedolisin family (MEROPS S53). This enzyme is active at low pH and in elevated temperatures. In this study that used x-ray crystallographic and biochemical methods, we investigated the structural basis of the preference of this enzyme for collagen and the importance of a glutamate residue in the unique catalytic triad (Ser(278)-Glu(78)-Asp(82)) for enzymatic activity. Crystal structures of the uninhibited enzyme and its complex with a covalently bound inhibitor, N-acetyl-isoleucyl-prolyl-phenylalaninal, showed the occurrence of a narrow S2 pocket and a groove that encompasses the active site and is rich in negative charges. Limited endoproteolysis studies of bovine type-I collagen as well as kinetic studies using peptide libraries randomized at P1 and P1', showed very strong preference for arginine at the P1 position, which correlated very well with the presence of a negatively charged residue in the S1 pocket of the enzyme. All of these features, together with those predicted through comparisons with fiddler crab collagenase, a serine peptidase, rationalize the enzyme's preference for collagen. A comparison of the Arrhenius plots of the activities of kumamolisin-As with either collagen or peptides as substrates suggests that collagen should be relaxed before proteolysis can occur. The E78H mutant, in which the catalytic triad was engineered to resemble that of subtilisin, showed only 0.01% activity of the wild-type enzyme, and its structure revealed that Ser(278), His(78), and Asp(82) do not interact with each other; thus, the canonical catalytic triad is disrupted.     

1.2 A crystal structure of the serine carboxyl proteinase pro-kumamolisin; structure of an intact pro-subtilase

Kumamolisin, an extracellular proteinase derived from an acido/thermophilic Bacillus, belongs to the sedolisin family of endopeptidases characterized by a subtilisin-like fold and a Ser-Glu-Asp catalytic triad. In kumamolisin, the Asp82 carboxylate hydrogen bonds to Glu32-Trp129, which might act as a proton sink stabilizing the catalytic residues. The 1.2/1.3 A crystal structures of the Glu32-->Ala and Trp129-->Ala mutants show that both mutations affect the active-site conformation, causing a 95% activity decrease. In addition, the 1.2 A crystal structure of the Ser278-->Ala mutant of pro-kumamolisin was determined. The prodomain exhibits a half-beta sandwich core docking to the catalytic domain similarly as the equivalent subtilisin prodomains in their catalytic-domain complexes. This pro-kumamolisin structure displays, for the first time, the uncleaved linker segment running across the active site and connecting the prodomain with the properly folded catalytic domain. The structure strongly points to an initial intramolecular activation cleavage in subtilases, as presumed for pro-subtilisin and pro-furin.     

Identification and analysis of a new family of bacterial serine proteinases

A family of hypothetical proteins, identified predominantly from archaeal genomes, has been analyzed in order to understand its functional characteristics. Using extensive sequence similarity searches it is inferred that this family is remotely related (best sequence identity is 19%) to ClpP proteinases that belongs to serine proteinase class. This family of hypothetical proteins is referred to as SDH proteinase family based on conserved sequential order of Ser, Asp and His residues and predicted serine proteinase activity. Results of fold recognition of SDH family sequences confirmed the remote relationship between SDH proteinases and Clp proteinases and revealed similar tertiary location of putative catalytic triad residues critical for serine proteinase function. However, the best sequence alignment we could obtain suggests that while catalytic Ser is conserved across Clp and SDH proteinases the location of the other catalytic triad residues, namely, His and Asp are swapped in their amino acid alignment positions and hence in 3-D structure. The evidence of conserved catalytic triad suggests that SDH could be a new family of serine proteinases with the fold of Clp proteinase, however sharing the catalytic triad order of carboxypeptidase clan. Signal peptide sequence identified at the N-terminus of some of the homologues suggests that these might be secretory serine proteinases involved in cleavage of extracellular proteins while the remote homologues, ClpP proteinases, are known to work in intracellular environment.     

Crystal structure and mechanism of tripeptidyl activity of prolyl tripeptidyl aminopeptidase from Porphyromonas gingivalis

The crystal structure of prolyl tripeptidyl aminopeptidase from Porphyromonas gingivalis was determined. Prolyl tripeptidyl aminopeptidase consists of beta-propeller and catalytic domains, and a large cavity between the domains; this structure is similar to dipeptidyl aminopeptidase IV. A catalytic triad (Ser603, His710, and Asp678) was located in the catalytic domain; this triad was virtually identical to that of the enzymes belonging to the prolyl oligopeptidase family. The structure of an inactive S603A mutant enzyme complexed with a substrate was also determined. The pyrrolidine ring of the proline residue appeared to fit into a hydrophobic pocket composed of Tyr604, Val629, Trp632, Tyr635, Tyr639, Val680, and Val681. There were characteristic differences in the residues of the beta-propeller domain, and these differences were related to the substrate specificity of tripeptidyl activity. The N-terminal amino group was recognized by salt bridges, with two carboxyl groups of Glu205 and Glu206 from a helix in dipeptidyl aminopeptidase IV. In prolyl tripeptidyl aminopeptidase, however, the Glu205 (located in the loop) and Glu636 were found to carry out this function. The loop structure provides sufficient space to accommodate three N-terminal residues (Xaa-Xaa-Pro) of substrates. This is the first report of the structure and substrate recognition mechanism of tripeptidyl peptidase.     

Identification of functionally important amino acid residues in Zymomonas mobilis levansucrase

The catalytic residues of levansucrase (sucrose:2,6-beta-D-fructan 6-beta-D-fructosyltransferase, EC 2.4.1.10) from Zymomonas mobilis were analyzed by random mutation and site-directed mutagenesis. We found that substitution of Glu278 with Asp and His reduced the k(cat) for sucrose hydrolysis 30- and 210-fold, respectively, strongly suggesting Glu278 plays a key role in catalyzing this reaction. Given the likelihood that another acidic amino residue was also involved, we constructed variants in which acidic amino acids located within homologous regions among bacterial levansucrases and fructosyltransferases were substituted, and found that substitution of Asp194, located in homologous region III, abolished sucrose hydrolysis. In addition, Glu278 was determined to be situated within the DXXER motif in homologous region IV conserved among bacterial levansucrases and fructosyltransferases, while Asp194 was within the triplet RDP motif conserved among bacterial levansucrases, fructosyltransferases and fructofuranosidases. Finally, comparison of our findings with published data on other site-directed mutated enzymes indicated His296, also located in homologous region IV, is crucial for catalysis of the transfructosylation reaction.     

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 catalytic site residues and interfacial binding of human pancreatic lipase.

In this study, the essential serine residue and 2 other amino acids in human pancreatic triglyceride lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) were tested for their contribution to the enzyme's catalytic site or interfacial binding site. By site-specific mutagenesis of the cDNA for human pancreatic lipase, amino acid substitutions were made at Ser153, His264, and Asp177. The mutant cDNAs were expressed in transfected COS-1 cells. Both the medium and the cells were examined for the presence of pancreatic lipase by Western blot analysis. The activity of the expressed proteins against triolein and the interfacial binding was measured. Proteins with mutations in Ser153 were secreted by the cells and bound to interfaces but had no detectable activity. Changing His264 to a leucine or Asp177 to an asparagine also produced inactive lipase. Substituting glutamic acid for Asp177 produced an active protein. These results demonstrate that Ser153 is involved in the catalytic site of pancreatic lipase and is not crucial for interfacial binding. Moreover, the essential roles of His264 and Asp177 in catalysis were demonstrated. A Ser-His-Asp catalytic triad similar to that present in serine proteases is present in human pancreatic lipase.     

Crystal structure of bovine duodenase, a serine protease, with dual trypsin and chymotrypsin-like specificities

The three-dimensional structure of duodenase, a serine protease from bovine duodenum mucosa, has been determined at 2.4A resolution. The enzyme, which has both trypsin-like and chymotrypsin-like activities, most closely resembles human cathepsin G with which it shares 57% sequence identity and similar specificity. The catalytic Ser195 in duodenase adopts the energetically favored conformation typical of serine proteinases and unlike the strained state typical of lipase/esterases. Of several waters in the active site of duodenase, the one associated with Ser214 is found in all serine proteinases and most lipase/esterases. The conservation of the Ser214 residue in serine proteinase, its presence in the active site, and participation in a hydrogen water network involving the catalytic triad (His57, Asp107, and Ser195) argues for its having an important role in the mechanism of action. It may be referred to as a fourth member of the catalytic triad. Duodenase is one of a growing family of enzymes that possesses trypsin-like and chymotrypsin-like activity. Not long ago, these activities were considered to be mutually exclusive. Computer modeling reveals that the S1 subsite of duodenase has structural features compatible with effective accommodation of P1 residues typical of trypsin (Arg/Lys) and chymotrypsin (Tyr/Phe) substrates. The determination of structural features associated with functional variation in the enzyme family may permit design of enzymes with a specific ratio of trypsin and chymotrypsin activities.     

Functional topology of a surface loop shielding the catalytic center in lipoprotein lipase.

Lipoprotein lipase (LPL), hepatic lipase, and pancreatic lipase show high sequence homology to one another. The crystal structure of pancreatic lipase suggests that it contains a trypsin-like Asp-His-Ser catalytic triad at the active center, which is shielded by a disulfide bridge-bounded surface loop that must be repositioned before the substrate can gain access to the catalytic residues. By sequence alignment, the homologous catalytic triad in LPL corresponds to Asp156-His241-Ser132, absolutely conserved residues, and the homologous surface loop to residues 217-238, a poorly conserved region. To verify these assignments, we expressed in vitro wild-type LPL and mutant LPLs having single amino acid mutations involving residue Asp156 (to His, Ser, Asn, Ala, Glu, or Gly), His241 (to Asn, Ala, Arg, Gln, or Trp), or Ser132 (to Gly, Ala, Thu, or Asp) individually. All 15 mutant LPLs were totally devoid of enzyme activity, while wild-type LPL and other mutant LPLs containing substitutions in other positions were fully active. We further replaced the 22-residue LPL loop which shields the catalytic center either partially (replacing 6 of 22 residues) or completely with the corresponding hepatic lipase loop. The partial loop-replacement chimeric LPL was found to be fully active, and the complete loop-replacement mutant had approximately 60% activity, although the primary sequence of the hepatic lipase loop is quite different. In contrast, replacement with the pancreatic lipase loop completely inactivated the enzyme. Our results are consistent with Asp156-His241-Ser132 being the catalytic triad in lipoprotein lipase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and amino acid sequence of a peptide containing an epoxide-reactive residue from the thermolysin-digest of Scytalidium lignicolum acid protease B

Scytalidium lignicolum acid protease B, a pepstatin-insensitive acid protease, was modified by 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) with the concomitant loss of its enzyme activity, and an EPNP-labeled peptide was isolated from the thermolysin-digest of the modified enzyme by HPLC. The amino acid sequence of the peptide was determined to be Ile-Leu-Glu-Thr-Gly, which corresponds to the sequence of residue Nos. 51-55 of the enzyme. The results of treatment of the labeled peptide with hydroxylamine suggested that the EPNP moiety is ester-linked to Glu53 of the enzyme. The amino acid sequence around Glu53 of the acid protease B showed high homology with those around the active site Asp residues of calf chymosin and porcine pepsin. These results show that it is highly possible that Glu53 of the acid protease B is one of the amino acid residues involved in its catalytic activity.     

Chlorophyllase as a serine hydrolase: identification of a putative catalytic triad

Chlorophyllases (Chlases), cloned so far, contain a lipase motif with the active serine residue of the catalytic triad of triglyceride lipases. Inhibitors specific for the catalytic serine residue in serine hydrolases, which include lipases effectively inhibited the activity of the recombinant Chenopodium album Chlase (CaCLH). From this evidence we assumed that the catalytic mechanism of hydrolysis by Chlase might be similar to those of serine hydrolases that have a catalytic triad composed of serine, histidine and aspartic acid in their active site. Thus, we introduced mutations into the putative catalytic residue (Ser162) and conserved amino acid residues (histidine, aspartic acid and cysteine) to generate recombinant CaCLH mutants. The three amino acid residues (Ser162, Asp191 and His262) essential for Chlase activity were identified. These results indicate that Chlase is a serine hydrolase and, by analogy with a plausible catalytic mechanism of serine hydrolases, we proposed a mechanism for hydrolysis catalyzed by Chlase.     

Characterization of the active site and thermostability regions of endoxylanase from Thermoanaerobacterium saccharolyticum B6A-RI.

Deletion mutants were constructed from pZEP12, which contained the intact Thermoanaerobacterium saccharolyticum endoxylanase gene (xynA). Deletion of 1.75 kb from the N-terminal end of xynA resulted in a mutant enzyme that retained activity but lost thermostability. Deletion of 1.05 kb from the C terminus did not alter thermostability or activity. The deduced amino acid sequence of T. saccharolyticum B6A-RI endoxylanase XynA was aligned with five other family F beta-glycanases by using the PILEUP program of the Genetics Computer Group package. This multiple alignment of amino acid sequences revealed six highly conserved motifs which included the consensus sequence consisting of a hydrophobic amino acid, Ser or Thr, Glu, a hydrophobic amino acid, Asp, and a hydrophobic amino acid in the catalytic domain. Endoxylanase was inhibited by EDAC [1-(3-dimethylamino propenyl)-3-ethylcarbodiimide hydrochloride], suggesting that Asp and/or Glu was involved in catalysis. Three aspartic acids, two glutamic acids, and one histidine were conserved in all six enzymes aligned. Hydrophobic cluster analysis revealed that two Asp and one Glu occur in the same hydrophobic clusters in T. saccharolyticum B6A-RI endoxylanase and two other enzymes belonging to family F beta-glycanases and suggests their involvement in a catalytic triad. These two Asp and one Glu in XynA from T. saccharolyticum were targeted for analysis by site-specific mutagenesis. Substitution of Asp-537 and Asp-602 by Asn and Glu-600 by Gln completely destroyed endoxylanase activity. These results suggest that these three amino acids form a catalytic triad that functions in a general acid catalysis mechanism.     

The complete amino acid sequence of rat submaxillary gland tonin does contain the aspartic acid at the active site: confirmation by protein sequence analysis

The revised amino acid sequence of rat submaxillary gland tonin, a serine protease, does contain the active site Asp residue. The active site of this kallikrein-related enzyme is thus made up of the same catalytic triad (Asp, Ser, and His) found in all known serine proteases. The important Asp residue has now been localized in a 16 amino acid peptide previously reported as missing in the tonin sequence. The complete amino acid sequence thus contains 235 residues corresponding to a molecular weight of 25,658, more in agreement with previously reported molecular weights. Moreover, the revised structure led (a) to the assignment of Arg, Asn, and Val residues instead of His, Asp, and Gly at positions 63, 165, and 169, respectively; (b) to the assignment of residues occupying an overlapping sequence at positions 165-171, and finally (c) to the localization of two N-glycosylation sites at positions 82 and 165. These results further document the close relationship of tonin to the ever expanding kallikrein family.     

Characterization of the iron- and 2-oxoglutarate-binding sites of human prolyl 4-hydroxylase.

Prolyl 4-hydroxylase (EC 1.14.11.2), an alpha2beta2 tetramer, catalyzes the formation of 4-hydroxyproline in collagens. We converted 16 residues in the human alpha subunit individually to other amino acids, and expressed the mutant polypeptides together with the wild-type beta subunit in insect cells. Asp414Ala and Asp414Asn inactivated the enzyme completely, whereas Asp414Glu increased the K(m) for Fe2+ 15-fold and that for 2-oxoglutarate 5-fold. His412Glu, His483Glu and His483Arg inactivated the tetramer completely, as did Lys493Ala and Lys493His, whereas Lys493Arg increased the K(m) for 2-oxoglutarate 15-fold. His501Arg, His501Lys, His501Asn and His501Gln reduced the enzyme activity by 85-95%; all these mutations increased the K(m) for 2-oxoglutarate 2- to 3-fold and enhanced the rate of uncoupled decarboxylation of 2-oxoglutarate as a percentage of the rate of the complete reaction up to 12-fold. These and other data indicate that His412, Asp414 and His483 provide the three ligands required for the binding of Fe2+ to a catalytic site, while Lys493 provides the residue required for binding of the C-5 carboxyl group of 2-oxoglutarate. His501 is an additional critical residue at the catalytic site, probably being involved in both the binding of the C-1 carboxyl group of 2-oxoglutarate and the decarboxylation of this cosubstrate.