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)     

Halide-stabilizing residues of haloalkane dehalogenases studied by quantum mechanic calculations and site-directed mutagenesis

Haloalkane dehalogenases catalyze cleavage of the carbon-halogen bond in halogenated aliphatic compounds, resulting in the formation of an alcohol, a halide, and a proton as the reaction products. Three structural features of haloalkane dehalogenases are essential for their catalytic performance: (i) a catalytic triad, (ii) an oxyanion hole, and (iii) the halide-stabilizing residues. Halide-stabilizing residues are not structurally conserved among different haloalkane dehalogenases. The level of stabilization of the transition state structure of S(N)2 reaction and halide ion provided by each of the active site residues in the enzymes DhlA, LinB, and DhaA was quantified by quantum mechanic calculations. The residues that significantly stabilize the halide ion were assigned as the primary (essential) or the secondary (less important) halide-stabilizing residues. Site-directed mutagenesis was conducted with LinB enzyme to confirm location of its primary halide-stabilizing residues. Asn38Asp, Asn38Glu, Asn38Phe, Asn38Gln, Trp109Leu, Phe151Leu, Phe151Trp, Phe151Tyr, and Phe169Leu mutants of LinB were constructed, purified, and kinetically characterized. The following active site residues were classified as the primary halide-stabilizing residues: Trp125 and Trp175 of DhlA; Asn38 and Trp109 of LinB; and Asn41 and Trp107 of DhaA. All these residues make a hydrogen bond with the halide ion released from the substrate molecule, and their substitution results in enzymes with significantly modified catalytic properties. The following active site residues were classified as the secondary halide-stabilizing residues: Phe172, Pro223, and Val226 of DhlA; Trp207, Pro208, and Ile211 of LinB; and Phe205, Pro206, and Ile209 of DhaA. The differences in the halide stabilizing residues of three haloalkane dehalogenases are discussed in the light of molecular adaptation of these enzymes to their substrates.     

Mutation analysis of carbamoyl phosphate synthetase: Does the structurally conserved glutamine amidotransferase triad act as a functional dyad?

Evolutionarily conserved triad glutamine amidotransferase (GAT) domains catalyze the cleavage of glutamine to yield ammonia and sequester the ammonia in a tunnel until delivery to a variety of acceptor substrates in synthetase domains of variable structure. Whereas a conserved hydrolytic triad (Cys/His/Glu) is observed in the solved GAT structures, the specificity pocket for glutamine is not apparent, presumably because its formation is dependent on the conformational change that couples acceptor availability to a greatly increased rate of glutamine cleavage. In Escherichia coli carbamoyl phosphate synthetase (eCPS), one of the best characterized triad GAT members, the Cys269 and His353 triad residues are essential for glutamine hydrolysis, whereas Glu355 is not critical for eCPS activity. To further define the glutamine-binding pocket and possibly identify an alternative member of the catalytic triad that is situated for this role in the coupled conformation, we have analyzed mutations at Gln310, Asn311, Asp334, and Gln351, four conserved, but not yet analyzed residues that might potentially function as the third triad member. Alanine substitution of Gln351, Asn311, and Gln310 yielded respective K(m) increases of 145, 27, and 15, suggesting that Gln351 plays a key role in glutamine binding in the coupled conformation, and that Asn311 and Gln310 make less significant contributions. None of the mutant k (cat) values varied significantly from those for wild-type eCPS. Combined with previously reported data on other conserved eCPS residues, these results strongly suggest that Cys269 and His353 function as a catalytic dyad in the GAT site of eCPS.     

Molecular cloning demonstrates structural features of homologous bovine prohormone convertases 1 and 2

PC1 and PC2 (prohormone convertase) represent neuroendocrine members of the mammalian subtilisin-like family of proprotein convertases. The goal of this study was to compare the primary sequence motifs of bovine PC1 and PC2 with those of homologs from other mammalian species to establish the structural basis for PC1 and PC2 activities in bovine that resemble other mammalian homologs. Molecular cloning from bovine adrenal medulla resulted in the isolation of cDNAs for bovine PC1 and PC2 with highly conserved primary sequences with respect to signal sequence, prosegment, catalytic domain, and P domain. Bovine PC1 and PC2 contained the catalytic triad residues Asp, His, Ser, which are identical to the triads in PC1 and PC2 from other mammalian species. Bovine PCl contained Asn as the oxyanion hole residue; in contrast, bovine PC2 contained Asp as the oxyanion hole residue, which is identical to PC2 in other mammalian species. Bovine PC1 and PC2 possessed the P domain that contains the functional RRGDL motif. The cloned cDNAs detected expression of PC1 and PC2 mRNAs in bovine adrenal medulla. These results establish the defined structural domains of bovine PC1 and PC2 that are known to be essential for the activities of these enzymes in various species.     

Function of the fully conserved residues Asp99, Tyr52 and Tyr73 in phospholipase A2

In the active centre of pancreatic phospholipase A2 His48 is at hydrogen-bonding distance to Asp99. This Asp-His couple is assumed to act together with a water molecule as a catalytic triad. Asp99 is also linked via an extended hydrogen bonding system to the side chains of Tyr52 and Tyr73. To probe the function of the fully conserved Asp99, Tyr52 and Tyr73 residues in phospholipase A2, the Asp99 residue was replaced by Asn, and each of the two tyrosines was separately replaced by either a Phe or a Gln. The catalytic and binding properties of the Phe52 and Phe73 mutants did not change significantly relative to the wild-type enzyme. This rules out the possibility that either one of the two Tyr residues in the wild-type enzyme can function as an acyl acceptor or proton donor in catalysis. The Gln73 mutant could not be obtained in any significant amounts probably due to incorrect folding. The Gln52 mutant was isolated in low yield. This mutant showed a large decrease in catalytic activity while its substrate binding was nearly unchanged. The results suggest a structural role rather than a catalytic function of Tyr52 and Tyr73. Substitution of asparagine for aspartate hardly affects the binding constants for both monomeric and micellar substrate analogues. Kinetic characterization revealed that the Asn99 mutant has retained no less than 65% of its enzymatic activity on the monomeric substrate rac 1,2-dihexanoyldithio-propyl-3-phosphocholine, probably due to the fact that during hydrolysis of monomeric substrate by phospholipase A2 proton transfer is not the rate-limiting step.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Structural basis for Ufm1 processing by UfSP1

Ubiquitin-fold modifier 1 (Ufm1) is a newly identified ubiquitin-like protein. Like ubiquitin and other ubiquitin-like proteins, Ufm1 is synthesized as a precursor that needs to be processed to expose the conserved C-terminal glycine prior to its conjugation to target proteins. Two novel proteases, named UfSP1 and UfSP2, have been shown to be responsible for the release of Ufm1 from Ufm1-conjugated cellular proteins as well as for the processing of its precursor. They show no sequence homology with known proteases. Here, we describe the 1.7A resolution crystal structure of mouse UfSP1, consisting of 217 amino acids. The structure reveals that it is a novel cysteine protease having a papain-like fold, with Cys(53), Asp(175), and His(177) that form a catalytic triad, and Tyr(41) that participates in the formation of the oxyanion hole. This differs from the canonical catalytic triad of papain-like proteases in that the aspartate and the histidine residues are from the "Asp-Pro-His" box. The Asp-Pro-His configuration seen in UfSP1, together with Atg4B and M48(USP), seem to form a new subfamily of the cysteine protease superfamily. The mutagenesis study of the active site residues confirms structural basis for catalysis. The interaction between UfSP1 and Ufm1 appears quite substantial, since the K(D) value was estimated to be 1.6 mum by the isothermal titration calorimetry analysis. Furthermore, the NMR data shows that the loop between beta3 and alpha2 in addition to the C-terminal region of Ufm1 plays a role in binding to UfSP1.     

Unusual catalytic triad of Escherichia coli outer membrane phospholipase A

Escherichia coli outer membrane phospholipase A (OMPLA) is an integral membrane enzyme. OMPLA is active as a homodimer and requires calcium as a cofactor. The crystal structures of the monomeric and the inhibited dimeric enzymes were recently determined [Snijder, H. J., et al. (1999) Nature 401, 717-721] and revealed that OMPLA monomers are folded into a 12-stranded antiparallel beta-barrel. The active site consists of previously identified essential residues Ser144 and His142 in an arrangement resembling the corresponding residues of a serine hydrolase catalytic triad. However, instead of an Asp or Glu that normally is present in the triad of serine hydrolases, a neutral asparagine (Asn156) was found in OMPLA. In this paper, the importance of the catalytic Asn156 is addressed by site-directed mutagenesis studies. All variants were purified at a 30 mg scale, and were shown to be properly folded using SDS-PAGE and circular dichroism spectroscopy. Using chemical cross-linking, it was shown that all variants were not affected in their calcium-dependent dimerization properties. The Asn156Asp variant exhibited a 2-fold lower activity than wild-type OMPLA at neutral pH. Interestingly, the activity of the variant is 1 order of magnitude higher than that of the wild type at pH >10. Modest residual activities (5 and 2.5%, respectively) were obtained for the Asn156Ala and Asn156Gln mutants, showing that the active site of OMPLA is more tolerant toward replacements of this third residue of the catalytic triad than other serine hydrolases, and that the serine and histidine residues are minimally required for catalysis. In the X-ray structure of dimeric OMPLA, the cofactor calcium is coordinating the putative oxyanion via two water molecules. We propose that this may lessen the importance for the asparagine in the catalytic triad of OMPLA.     

New insight into the architecture of oxy‐anion pocket in unliganded conformation of GAT domains: A MD‐simulation study

Human Guanine Monophosphate Synthetase (hGMPS) converts XMP to GMP, and acts as a bifunctional enzyme with N‐terminal “glutaminase” (GAT) and C‐terminal “synthetase” domain. The enzyme is identified as a potential target for anti‐cancer and immunosuppressive therapies. GAT domain of enzyme plays central role in metabolism, and contains conserved catalytic residues Cys104, His190, and Glu192. MD simulation studies on GAT domain suggest that position of oxyanion in unliganded conformation is occupied by one conserved water molecule (W1), which also stabilizes that pocket. This position is occupied by a negatively charged atom of the substrate or ligand in ligand bound crystal structures. In fact, MD simulation study of Ser75 to Val indicates that W1 conserved water molecule is stabilized by Ser75, while Thr152, and His190 also act as anchor residues to maintain appropriate architecture of oxyanion pocket through water mediated H‐bond interactions. Possibly, four conserved water molecules stabilize oxyanion hole in unliganded state, but they vacate these positions when the enzyme (hGMPS)‐substrate complex is formed. Thus this study not only reveals functionally important role of conserved water molecules in GAT domain, but also highlights essential role of other non‐catalytic residues such as Ser75 and Thr152 in this enzymatic domain. The results from this computational study could be of interest to experimental community and provide a testable hypothesis for experimental validation. Conserved sites of water molecules near and at oxyanion hole highlight structural importance of water molecules and suggest a rethink of the conventional definition of chemical geometry of inhibitor binding site. Proteins 2016; 84:360–373. © 2016 Wiley Periodicals, Inc.     

Analysis of the reaction mechanism and substrate specificity of haloalkane dehalogenases by sequential and structural comparisons

Haloalkane dehalogenases catalyse environmentally important dehalogenation reactions. These microbial enzymes represent objects of interest for protein engineering studies, attempting to improve their catalytic efficiency or broaden their substrate specificity towards environmental pollutants. This paper presents the results of a comparative study of haloalkane dehalogenases originating from different organisms. Protein sequences and the models of tertiary structures of haloalkane dehalogenases were compared to investigate the protein fold, reaction mechanism and substrate specificity of these enzymes. Haloalkane dehalogenases contain the structural motifs of alpha/beta-hydrolases and epoxidases within their sequences. They contain a catalytic triad with two different topological arrangements. The presence of a structurally conserved oxyanion hole suggests the two-step reaction mechanism previously described for haloalkane dehalogenase from Xanthobacter autotrophicus GJ10. The differences in substrate specificity of haloalkane dehalogenases originating from different species might be related to the size and geometry of an active site and its entrance and the efficiency of the transition state and halide ion stabilization by active site residues. Structurally conserved motifs identified within the sequences can be used for the design of specific primers for the experimental screening of haloalkane dehalogenases. Those amino acids which were predicted to be functionally important represent possible targets for future site-directed mutagenesis experiments.     

Mutational analysis of Cvab, an ABC transporter involved in the secretion of active colicin V

CvaB is the central membrane transporter of the colicin V secretion system that belongs to an ATP-binding cassette superfamily. Previous data showed that the N-terminal and C-terminal domains of CvaB are essential for the function of CvaB. N-terminal domain of CvaB possesses Ca(2+)-dependent cysteine proteolytic activity, and two critical residues, Cys32 and His105, have been identified. In this study, we also identify Asp121 as being the third residue of the putative catalytic triad within the active site of the enzyme. The Asp121 mutants lose both their colicin V secretion activity and N-terminal proteolytic activity. The adjacent residue Pro122 also appears to play a critical role in the colicin V secretion. However, the reversal of the two residues D121P - P122D results in loss of activity. Based on molecular modeling and protein sequence alignment, several residues adjacent to the critical residues, Cys32 and His105, were also examined and characterized. Site-directed mutagenesis of Trp101, Asp102, Val108, Leu76, Gly77, and Gln26 indicate that the neighboring residues around the catalytic triad affect colicin V secretion. Several mutated CvaB proteins with defective secretion were also tested, including Asp121 and Pro122, and were found to be structurally stable. These results indicate that the residues surrounding the identified catalytic triad are functionally involved in the secretion of biologically active colicin V.     

Consequences of breaking the Asp-His hydrogen bond of the catalytic triad: effects on the structure and dynamics of the serine esterase cutinase.

The objective of this study has been to investigate the effects on the structure and dynamics that take place with the breaking of the Asp-His hydrogen bond in the catalytic triad Asp175-His188-Ser120 of the serine esterase cutinase in the ground state. Four molecular dynamics simulations were performed on this enzyme in solution. The starting structures in two simulations had the Asp175-His188 hydrogen bond intact, and in two simulations the Asp175-His188 hydrogen bond was broken. Conformations of the residues comprising the catalytic triad are well behaved during both simulations containing the intact Asp175-His188 hydrogen bond. Short contacts of less than 2.6 A were observed in 1.2% of the sampled distances between the carboxylate oxygens of Asp175 and the NE2 of His188. The simulations showed that the active site residues exhibit a great deal of mobility when the Asp175-His188 hydrogen bond is broken. In the two simulations in which the Asp175-His188 hydrogen bond is not present, the final geometries for the residues in the catalytic triad are not in catalytically productive conformations. In both simulations, Asp175 and His188 are more than 6 A apart in the final structure from dynamics, and the side chains of Ser120 and Asp175 are in closer proximity to the NE2 of His188 than to ND1. Nonlocal effects on the structure of cutinase were observed. A loop formed by residues 26-31, which is on the opposite end of the protein relative to the active site, was greatly affected. Further changes in the dynamics of cutinase were determined from quasiharmonic mode analysis. The frequency of the second lowest mode was greatly reduced when the Asp175-His188 hydrogen bond was broken, and several higher modes showed lower frequencies. All four simulations showed that the oxyanion hole, composed of residues Ser42 and Gln121, is stable. Only one of the hydrogen bonds (Ser42 OG to Gln121 NE2) observed in the crystal structure that stabilize the conformation of Ser42 OG persisted throughout the simulations. This hydrogen bond appears to be enough for the oxyanion hole to retain its structural integrity.     

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.     

Homology modelling and site-directed mutagenesis studies of the epoxide hydrolase from Phanerochaete chrysosporium

Three-dimensional structural model of epoxide hydrolase (PchEHA) from Phanerochaete chrysosporium was constructed based on X-ray structure of Agrobacterium radiobacter AD1 sEH using SWISS-MODEL server. Conserved residues constituting the active site cavity were identified, of which the functional roles of 14 residues were determined by site-directed mutagenesis. In catalytic triad, Asp105 and His308 play a leading role in alkylation and hydrolysis steps, respectively. Distance between Asp105 and epoxide ring of substrate may determine the regiospecificity in the substrate docking model. Asp277 located at the entrance of substrate tunnel is concerned with catalysis but not essential. D307E had the highest activity and lower enantioselectivity among 14 mutants, suggesting Asp307 may be involved in choice of substrate configuration. Y159F and Y241F almost exhibited no activity, indicating that they are essential to bind substrate and facilitate opening of epoxide ring. Besides, His35-Gly36-Asn37-Pro38, Trp106 and Trp309 surrounding Asp105, may coordinate the integration of active site cavity and influence substrate binding. Especially, W106I reversed the enantioselectivity, perhaps due to more deteriorative impact on the preferred (R)-styrene oxide. Gly65 and Gly67 occurring at β-turns and Gly36 are vital in holding protein conformation. Conclusively, single conserved residue around the active sites has an important impact on catalytic properties.     

Role of catalytic residues in the formation of a tetrahedral adduct in the acylation reaction of bovine β-trypsin: A molecular orbital study

In the acylation reaction of serine proteases the effect of amino acid residues on the geometrical change of the catalytic site from Michaelis to tetrahedral state was studied by using ab initio molecular orbital calculations. Amino acid residues in the catalytic site and the peptide substrate were calculated as a quantum mechanical region, and all the other amino acid residues and the calcium ion were included in the calculation as the electrostatic effects. The effects of Asp102, Asp194, N-terminus and the oxyanion binding site are large. The oxyanion binding site directly stabilizes the tetrahedral substrate. Asp102 stabilizes the enzyme intermediate, interacting with the protonated His57 residue. In order to elucidate the roles of Asp102 and the oxyanion binding site, energy decomposition analyses were done for the intermolecular interactions. The contribution of Asp102 and the oxyanion binding site to the decrease of energy in the geometrical change is due to the electrostatic effect. The energies of the proton shuttle from Ser195 O^γ to the leaving group of the substrate were calculated for amide and ester substrate models.     

Identification and characterisation of the catalytic triad of the alkaliphilic thermotolerant PHA depolymerase PhaZ7 of Paucimonas lemoignei

The recently discovered extracellular poly[(R)-3-hydroxybutyrate] (PHB) depolymerase PhaZ7 of Paucimonas lemoignei represents the first member of a new subgroup (EC 3.1.1.75) of serine hydrolases with no significant amino acid similarities to conventional PHB depolymerases, lipases or other hydrolases except for a potential lipase box-like motif (Ala-His-Ser136-Met-Gly) and potential candidates for catalytic triad and oxyanion pocket amino acids. In order to identify amino acids essential for activity 11 mutants of phaZ7 were generated by site-directed mutagenesis and expressed in recombinant protease-deficient Bacillus subtilis WB800. The wild-type depolymerase and 10 of the 11 mutant proteins (except for Ser136Cys) were expressed and efficiently secreted by B. subtilis as shown by Western blots of cell-free culture fluid proteins. No PHB depolymerase activity was detected in strains harbouring one of the following substitutions: His47Ala, Ser136Ala, Asp242Ala, Asp242Asn, His306Ala, indicating the importance of these amino acids for activity. Replacement of Ser136 by Thr resulted in a decrease of activity to about 20% of the wild-type level and suggested that the hydroxy group of the serine side chain is important for activity but can be partially replaced by the hydroxy function of threonine. Alterations of Asp256 to Ala or Asn or of the putative serine hydrolase pentapeptide motif (Ala-His-Ser136-Met-Gly) to a lipase box consensus sequence (Gly134-His-Ser136-Met-Gly) or to the PHB depolymerase box consensus sequence (Gly134-Leu135-Ser136-Met-Gly) had no significant effect on PHB depolymerase activity, indicating that these amino acids or sequence motifs were not essential for activity. In conclusion, the PHB depolymerase PhaZ7 is a serine hydrolase with a catalytic triad and oxyanion pocket consisting of His47, Ser136, Asp242 and His306.     

Comparison between catalase-peroxidase and cytochrome c peroxidase. The role of the hydrogen-bond networks for protein stability and catalysis

A detailed resonance Raman and electronic absorption investigation has been carried out on a series of novel distal and proximal variants of recombinant catalase-peroxidase from the cyanobacterium Synechocystis PCC 6803. In particular, variants of the distal triad Pro-Asp-Asn and the proximal triad His-Asp-Trp have been studied in their ferric and ferrous states at various pH. The data suggest marked differences in the structural role of the conserved residues and hydrogen-bond networks in KatG and CCP, which might be connected to the different catalytic activity. In particular, in KatG the proximal residues have a major role in the stability of the protein architecture because the disruption of the proximal Trp-Asp hydrogen bond by mutation weakens heme binding to the protein. On the distal side, replacing the hydrogen-acceptor carboxamide group of Asn153 by an aspartate carboxylate group or an aliphatic residue alters or disrupts the hydrogen bond with the distal His. As a consequence, the basicity of His123 is altered. The effect of mutation on Asp152 is noteworthy. Replacement of the Asp152 with Ser makes the architecture of the protein very similar to that of CCP. The Asp152 residue, which has been shown to be important in the hydrogen peroxide oxidation reaction, is expected to be hydrogen bonded to the nitrogen atom of Ile248 which is part of the KatG-specific insertion LL1, as in other KatGs. This insertion is at one edge of the heme, and connects the distal side with the proximal helices E and F, the latter carrying the proximal His ligand. We found that the distal Asp-Ile hydrogen bond is important for the stability of the heme architecture and its alteration changes markedly the proximal His-Asp hydrogen-bond interaction.     

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.     

A structural model for the prostate disease marker, human prostate-specific antigen.

Prostate-specific antigen (PSA) provides an excellent serum marker for prostate cancer, the most frequent form of cancer in American males. PSA is a 237-residue protease based on sequence homology to kallikrein-like enzymes. To predict the 3-dimensional structure of PSA, homology modeling studies were performed based on sequence and structural alignments with tonin, pancreatic kallikrein, chymotrypsin, and trypsin. The structurally conserved regions of the 4 reference X-ray proteins provided the core structure of PSA, whereas the loop structures were modeled on the loops of tonin and kallikrein. The unique "kallikrein loop" insert, between Ser 95b and Pro 95k of kallikrein, was constructed using molecular mechanics, dynamics, and electrostatics calculations. In the resulting PSA structure, the catalytic triad, involving residues His 57, Asp 102, and Ser 195, and hydrophobic and electrostatic interactions typical of serine proteases were extremely well conserved. Similarly, the 5-disulfide bonds of kallikrein were also conserved in PSA. These results, together with the fact that no major steric clashes arose during the modeling process, provide strong evidence for the validity of the PSA model. Calculation of the electrostatic potential contours of kallikrein and PSA was carried out using the finite difference Poisson-Boltzmann method. The calculations revealed matching areas of negative potential near the catalytic triad, but differences in the positive potential surrounding the active site. The PSA glycosylation site, Asn 61, is fully accessible to the solvent and is enclosed in a positive region of the isopotential map. The bottom of the substrate specificity pocket, residue S1, is a serine (Ser 189) as in chymotrypsin, rather than aspartate (Asp 189) as in tonin, kallikrein, and trypsin. This fact, plus other features of the S1 binding-pocket region, suggest that PSA would prefer substrates with hydrophobic residues at the P1 position. The location of a potential zinc ion binding site involving the side chain of histidines 91, 101, and 233 is also suggested. This PSA model should facilitate the understanding and prediction of structural and functional properties of this important cancer marker.