Catalytic Triad Of Serine Proteases Research Articles

Pen c 1, a novel enzymic allergen protein from Penicillium citrinum. Purification, characterization, cloning and expression.

A 33-kDa alkaline serine protease secreted by Penicillium citrinum strain 52-5 is shown to be an allergenic agent in this fungus. The protein, designated Pen c 1, was purified by sequential DEAE-Sepharose and carboxymethyl (CM)-Sepharose chromatographies. Pen c 1 has a molecular mass of 33 kDa and a pI of 7.1. The caseinolytic enzyme activity of this protein was studied. The protein binds to serum IgE from patients allergic to Penicillium citrinum. The cDNA encoding Pen c 1 is 1420 bp in length and contains an open reading frame for a 397-amino-acid polypeptide. Pen c 1 codes for a larger precursor containing a signal peptide, a propeptide and the 33-kDa mature protein. Sequence comparison revealed that Pen c 1 possesses several features in common with the alkaline serine proteases of the subtilisin family. The essential Asp, His, and Ser residues that make up the catalytic triad of serine proteases are well conserved. Northern blots demonstrated that mRNAs transcribed from this gene are present at early stages of culture. The allergen encoded by Pen c 1 gene was expressed in Escherichia coli as a fusion protein bearing an N-terminal histidine-affinity tag. The protein, purified by affinity chromatography with a yield of 130 mg of pure protein per liter of culture, was able to bind to both a monoclonal anti-Pen c 1 antibody and IgE from the serum of patients allergic to Penicillium. Recombinant Pen c 1 can therefore be expressed in E. coli in large quantities and should prove useful as a standardized specific allergen for immuno-diagnosis of atopic disorders. In addition, full caseinolytic enzyme activity could be generated in the purified recombinant protein by sulfonation and renaturation, followed by removal of the affinity tag, indicating that the refolded protein can assume the same conformation as the native protein.

Molecular cloning and characterization of prostase, an androgen-regulated serine protease with prostate-restricted expression

The identification of genes with selective expression in specific organs or cell types provides an entry point for understanding biological processes that occur uniquely within a particular tissue. Using a subtraction approach designed to identify genes preferentially expressed in specific tissues, we have identified prostase, a human serine protease with prostate-restricted expression. The prostase cDNA encodes a putative 254-aa polypeptide with a conserved serine protease catalytic triad and an amino-terminal pre-propeptide sequence, indicating a potential secretory function. The genomic sequence comprises five exons and four introns and contains multiple copies of a chromosome 19q-specific minisatellite repeat. Northern analysis indicates that prostase mRNA is expressed in hormonally responsive normal and neoplastic prostate epithelial tissues, but not in prostate stromal constituents. Prostase shares 35% amino acid identity with prostate-specific antigen (PSA) and 78% identity with the porcine enamel matrix serine proteinase 1, an enzyme involved in enamel matrix degradation and with a putative role in the disruption of intercellular junctions. Radiation-hybrid-panel mapping localized prostase to chromosome 19q13, a region containing several other serine proteases, including protease M, pancreatic/renal kallikrein hK1, and the prostate-specific kallikreins hK2 and hK3 (PSA). The sequence homology between prostase and other well-characterized serine proteases suggests several potential functional roles for the prostase protein that include the degradation of extracellular matrix and the activation of PSA and other proteases.

Theoretical evaluation of a model of the catalytic triads of serine and cysteine proteases by ab initio molecular orbital calculation.

Serine and cysteine proteases are structurally distinct families of proteolytic enzymes that exert analogous catalytic reactions. It is known that serine, histidine, and aspartate residues are involved in a putative catalytic triad of serine proteases whereas cysteine, histidine and aspartate (or asparagine) are involved in that of cysteine proteases. Although the overall three-dimensional structures of the two classes of proteases are quite different, a similar feature of the catalytic mechanism can be observed. We carried out a theoretical study in the gas phase on their two catalytic steps using ab initio molecular orbital calculation to evaluate the efficiency of each proteolytic activity. To examine the key aspects of the catalytic processes in their active sites, we employed simple molecular models, in which serine and histidine were substituted for by CH3OH and CH3SH, respectively, and aspartate by CH3CO2-. Molecular geometries of the two models were fully optimized by an energy-gradient method. The activation energies for proton transfer in the proteolytic steps and the total energies of each molecule in the intermediate states were calculated. These results revealed that the activation energy for proton transfer in the CH3SH-His-CH3CO2-system was smaller by 11 kcal mol-1than that of the CH3OH-His-CH3CO2-system. The calculated potential energy surface and the relative magnitudes of all steps in these reaction paths with regard to double proton transfer supported the above results. Taken together, these results indicated that the Cys-His-Asp proteolytic system might work more efficiently than the Ser-His Asp system.

Cloning and Tissue Distribution of a Novel Serine Protease esp-1 from Human Eosinophils

We have cloned a novel serine protease designated as esp-1 from human eosinophils. The amino acid sequence deduced from the cDNA showed that ESP-1 comprises a signal peptide of 18 amino acids, a propeptide of 23 amino acids, an active form sequence of 273 amino acids starting from an Ile-Val-Gly-Gly-Glu motif, the catalytic triad of serine proteases that has been characterized as the essential amino acid residues for the proteolytic activity, and a hydrophobic amino acid stretch in the carboxyl terminus, suggesting this enzyme is a novel membrane-type serine protease. The tissue distributions of esp-1 expression revealed that this protease is not only expressed in human eosinophils, but also widely expressed in mononuclear cells and various tissues other than skeletal muscle and kidney and is most abundant in testis and prostate, and moderately so in lung, spleen and pancreas.

Quantum chemical calculations on the intramolecular hydrogen bond of cis-urocanic acid

The intramolecular hydrogen bond (IMHB) of the neutral, cationic, and anionic cis-urocanic acid ( cis-UCA, (Z)-3-(I′ H-imidazol-4′(5′)-yl)propenoic acid) is studied using ab initio calculations up to the MP2/6-31+G** level of theory. The IMHB energies are estimated by first showing a near to perfect linear correlation between the hydrogen bond energies of modified cis-urocanic acid structures and the Mulliken bond orders of their hydrogen bonds — the electron densities at the critical points of the hydrogen bonds have a weaker correlation with the hydrogen bond energies than the bond orders — and then using this correlation in order to evaluate the IMHB energies of the cis-UCA structures themselves. This method gives high (>50 kJ mol −1) IMHB energies for the neutral and cationic cis-UCA structures and very high (>100 kJ mol −1) IMHB energies for the anionic cis-UCA structures in the gas phase. The effect of salvation on the IMHB in cis-UCA is studied using both MP2/6-31G* water–UCA supermolecule optimizations and HF/6-31G* SCI-PCM (self-consistent isodensity polarized continuum model) reaction field calculations. Solvation changes the IMHB energies by upto 30 kJ mol −1. The IMHB of the imidazole protonated structures is weakened, while that of the carboxylate group protonated structures is strengthened in aqueous solution. Solvation does not change the optimal conformation of cis-UCA from the IMHB-ed closed conformation to an open conformation. The supermolecule calculations and the SCI-PCM calculations give consistent changes of the IMHB strengths in aqueous solution. Inclusion of only 1–3 water molecules nearest to the IMHB seems to be enough to demonstrate most of the solvent effect on the IMHB. The semiempirical AM l method is shown to be a valuable tool in finding good initial positions for the water molecules in ab initio calculations. The adequacy of cis-UCA as a model compound for the hydrogen bonded His–Asp component of the catalytic triad in serine proteases is investigated. The IMHB in the anionic cis-UCA is a low-barrier hydrogen bond (LBHB) in the gas phase, while the IMHB in the neutral cis-UCA is not. In aqueous solution, modeled by HF/6-31G* SCI-PCM calculations, the IN4HB in both the neutral and the anionic cis-UCA seems to be an LBHB. The anionic cis-UCA is suggested as an experimental model for the initial state, and the neutral cis-UCA as that for the final state of the proton transfer process in the His–Asp system of the catalytic triad

The 0.78 A structure of a serine protease: Bacillus lentus subtilisin.

Ultrahigh-resolution X-ray diffraction data from cryo-cooled, B. lentus subtilisin crystals has been collected to a resolution of 0.78 A. The refined model coordinates have a rms deviation of 0.22 A relative to the same structure determined at room temperature and 2.0 A resolution. Several regions of main-chain and side-chain disorder have been identified for 21 out of 269 residues in one polypeptide chain. Hydrogen atoms appear as significant peaks in the Fo - Fc difference electron density map, and carbon, nitrogen, and oxygen atoms can be differentiated. The estimated standard deviation (ESD) for all main-chain non-hydrogen bond lengths is 0.009 A and 0.5 degrees for bond angles based on an unrestrained full-matrix least-squares refinement. Hydrogen bonds are resolved in the serine protease catalytic triad (Ser-His-Asp). Electron density is observed for an unusual, short hydrogen bond between aspartic acid and histidine in the catalytic triad. The hydrogen atom, identified by NMR in numerous serine proteases, appears to be shared by the heteroatoms in the bond. This represents the first reported correlation between detailed chemical features identified by NMR and those in a cryo-cooled crystallographic structure determination at ultrahigh resolution. The short hydrogen bond, designated "catalytic hydrogen bond", occurs as part of an elaborate hydrogen bond network, involving Asp of the catalytic triad. While unusual, these features appear to have conserved analogues in other serine protease families although specific details differ from family to family.

Identification and Characterization of DegP, a Serine Protease Associated with the Luminal Side of the Thylakoid Membrane

The proteases involved in proteolytic degradation in the thylakoid lumen are largely unknown. Western analysis with an antibody against the Escherichia coli periplasmic serine protease DegP suggested that pea chloroplasts contain a homologue of this protease. This homologue was peripherally bound to the luminal side of the thylakoid membrane and could only be removed by a combination of high salt and non-ionic detergent. Its level increased almost 2-fold in pea seedlings exposed to elevated temperature for 4 h, suggesting this protease's role in the chloroplast's heat response. Isolated thylakoid membranes containing the chloroplastic homologue of DegP degraded beta-casein, an in vitro substrate of the bacterial protease. This activity was partially inhibited by a serine protease inhibitor, suggesting that at least part of the casein-degrading activity in the thylakoid membrane is attributable to DegP. The existence of chloroplastic DegP was further supported by isolating a full-length Arabidopsis cDNA (designated AtDegP) encoding a protein that is 37% identical and 60% similar to the E. coli protease. The amino terminus of the deduced amino acid sequence contained a bipartite transit peptide, typical of proteins targeted to the thylakoid lumen, and the mature portion of the protein contained the highly conserved serine protease catalytic triad His-Asp-Ser. The possible physiological roles of chloroplastic DegP protease are discussed.

Linkers of secondary structures in proteins.

Linkers that connect repeating secondary structures fall into conformational classes based on distance and main-chain torsion clustering. A data set of 300 unique protein chains with low pairwise sequence identity was clustered into only a few groups representing the preferred motifs. The linkers of two to eight residues for the nonredundant data set are designated H-Ln-H, H-Ln-E, E-Ln-H, E-Ln-E, where n is the length, H stands for alpha-helices, and E for beta-strands. Most of the clusters identified here corroborate earlier findings. However, 19 new clusters are identified in this paper, with many of them having seven and eight residue linkers. In our first analysis, the secondary structures flanking the linkers are both interacting and noninteracting and there is no precise angle of orientation between them. A second analysis was performed on a set of proteins with restricted orientations for the flanking elements, namely, mainly alpha class of proteins with orthogonal architecture. Two definite clusters are identified, one corresponding to linkers of orthogonal helices and the other to linkers of antiparallel helices. Loops forming binding sites or involved in catalytic activity are important determinants of the function of proteins. Although the structural conservation of the residues around the catalytic triad of serine proteases has been studied widely, there has not been a systematic analysis of the conformation of the loops that contain them. Residues of the catalytic triad reside in the linkers of beta-strands, with varying lengths of more than eight residues. Here, we analyze the structural conservation of such linkers by superposition, and observe a conserved structural feature of the linkers incorporating each of the three residues of the catalytic triad.

A low-barrier hydrogen bond in the catalytic triad of serine proteases? Theory versus experiment.

Cleland and Kreevoy recently advanced the idea that a special type of hydrogen bond (H-bond), termed a low-barrier hydrogen bond (LBHB), may account for the "missing" transition state stabilization underlying the catalytic power of many enzymes, and Frey et al. have proposed that the H-bond between aspartic acid 102 and histidine 57 in the catalytic triad of serine proteases is an example of a catalytically important LBHB. Experimental facts are here considered regarding the aspartic acid-histidine and cis-urocanic H-bonds that are inconsistent with fundamental tenets of the LBHB hypothesis. The inconsistencies between theory and experiment in these paradigm systems cast doubt on the existence of LBHBs, as currently defined, within enzyme active sites.

Cloning and sequencing of two genes, prtA and prtB, from Myxococcus xanthus, encoding PrtA and PrtB proteases, both of which are required for the protease activity

The sequence of a 1955-bp TaqI DNA fragment from Myxococcus xanthus was determined. This fragment contains two complete genes, designated prtA and prtB. The prtA and prtB ORFs extend over 828 and 798 bp, respectively. They are separated only by 3 nt and appear to be present in a polycistronic transcriptional unit. A typical lipoprotein signal sequence is present at the N terminus of the two deduced polypeptides. The aa sequence of PrtA shows a high degree of identity to the region adjacent to the Ser residue belonging to the catalytic triad of serine proteases from Staphylococcus aureus and Enterococcus faecalis. It also exhibits features characteristic of trypsin-like serine proteases in that it contains the same pattern of variable and conserved regions. The deduced aa sequence of PrtB reveals a signature zinc-binding consensus motif (HEXXHXXGXXH/Met-turn) characteristic of the class of metalloproteases called metzincins. Plasmids containing prtA, prtB, or both were constructed. Protease activity studies of Escherichia coli clones containing these plasmids showed that both genes are necessary for this activity, whatever their cis or trans position. As prtB produces a putative membrane-bound lipoprotein of 266 aa, the protease activation must occur at the membrane level.

CDNA cloning and pattern of expression of an adult, female-specific chymotrypsin from Aedes aegypti midgut

A cDNA for a midgut chymotrypsin, induced by a blood meal, has been cloned and sequenced from the mosquito Aedes aegypti. The 938 base sequence codes for a 268 amino acid protein, which contains an 18-residue signal peptide and a seven-residue activation peptide. The deduced amino acid sequence contains several features typical of chymotrypsin proteases, including the catalytic triad of serine proteases and the residues that determine the chymotrypsin substrate specificity pocket. The chymotrypsin mRNA, absent in larvae, pupae, males and newly emerged females, reaches detectable levels within 24 h post-emergence and attains a maximum level 3–7 days after emergence. Translation of the chymotrypsin mRNA is induced by feeding a protein meal, and there is a dramatic increase in midgut chymotrypsin enzymatic activity after feeding. Chymotrypsin activity remained high during protein digestion, but chymotrypsin protein levels and enzymatic activity were almost undetectable once digestion was completed, 48 h after feeding.

Structure of the Human Cytomegalovirus Protease Catalytic Domain Reveals a Novel Serine Protease Fold and Catalytic Triad

Proteolytic processing of capsid assembly protein precursors by herpesvirus proteases is essential for virion maturation. A 2.5 Å crystal structure of the human cytomegalovirus protease catalytic domain has been determined by X-ray diffraction. The structure defines a new class of serine protease with respect to global-fold topology and has a catalytic triad consisting of Ser-132, His-63, and His-157 in contrast with the Ser-His-Asp triads found in other serine proteases. However, catalytic machinery for activating the serine nucleophile and stabilizing a tetrahedral transition state is oriented similarly to that for members of the trypsin-like and subtilisin-like serine protease families. Formation of the active dimer is mediated primarily by burying a helix of one protomer into a deep cleft in the protein surface of the other.

A low-barrier hydrogen bond in subtilisin: 1H and 15N NMR studies with peptidyl trifluoromethyl ketones.

The N delta 1 proton of His 64 forms a hydrogen bond with Asp 32, as part of the catalytic triad in serine proteases of the subtilisin family. His 64 in subtilisin has been studied by 1H and 15N NMR spectroscopy in the presence and absence of peptidyl trifluoromethyl ketones (TFMKs) that are transition state analog inhibitors. For subtilisin Carlsberg, the downfield resonance of the imidazolium N delta 1 proton is approximately 18.3 ppm and the D/H fractionation factor is 0.55 +/- 0.04 at pH 5.5 (11 degrees C), and 0.63 +/- 0.04 (5 degrees C) and 0.68 +/- 0.04 at pH 6 (11 degrees C). In the complex between subtilisin Carlsberg and Z-L-leucyl-L-leucyl-L-phenylalanyltrifluoromethyl ketone (Z-LLF-CF3) at pH values between 6.5 and 10.6, His 64 remains positively charged, and the D/H fractionation factor of its N delta 1 proton is 0.85 +/- 0.05. In the complex between a subtilisin variant from Bacillus lentus and Z-LLF-CF3, the proton resonance at 18.8 ppm is correlated with a 15N resonance at 197.6 ppm downfield from liquid NH3 with a 1JNH of 81 Hz. The chemical shifts of subtilisin complexes with peptidyl TFMKs are among the most downfield shifts reported for any protein. At pH 9.5, His 64 is neutral and the D/H fractionation factor increases to 1.2 with a chemical shift of 15.0. His 64 is positively charged in the free enzyme at low pH, the inhibitor hemiketal complex at neutral pH, and the transition state for amide bond hydrolysis. These data thus provide indirect evidence for the presence of a low-barrier hydrogen bond in the catalytic mechanism of subtilisin proteases.

Molecular Cloning and Characterization of Anionic and Cationic Variants of Trypsin from Atlantic Salmon

Pancreatic cDNA libraries from Atlantic salmon (Salmo salar) were constructed and screened with salmon trypsin-specific probes. Five clones containing near full-length transcripts were selected for further characterization. Comparison of deduced amino acid sequences revealed that all variants possessed the canonical serine protease catalytic triad, consisting of histidine, aspartic acid and serine residues, a substrate-binding pocket with aspartic acid at the bottom, and 12 cysteine residues comprising six disulphide bridges. Translation in vitro of one of the trypsin clones produced a protein with the expected molecular mass of 24.5 kDa. Three of the Atlantic salmon trypsins (SalTRP-I, SalTRP-IA and SalTRP-IB) possessed very similar sequences and may represent allelic variants encoded by the same gene focus; however, existence as tetraploid loci or isoloci where disomic inheritance is incomplete may also exist in Atlantic salmon and cannot be excluded. Two other trypsin clones (SalTRP-II and SalTRP-III) are probably encoded by separate gene loci. Analysis of genomic DNA by Southern blotting and hybridization to a trypsin probe showed a complex pattern, indicative of a large number of gene loci for trypsin in Atlantic salmon. The charged amino acid distribution showed that four of the Atlantic salmon trypsin clones encoded anionic forms of the enzyme, while the fifth clone represented a cationic variant. Multiple alignments of the Atlantic salmon trypsin sequences with trypsin, chymotrypsin and elastase from different species placed all Atlantic salmon sequences approximately equidistant from trypsins of other species. Interestingly, the distance between the anionic and cationic variants from Atlantic salmon was similar to the distance between salmon and mammalian trypsins, revealing an early separation of these two types of trypsin, possibly prior to the derivation of fish during evolution. A structural model based on X-ray diffraction studies of the salmon trypsin protein was very similar to that of the mammalian enzyme. All residues which differ in charge between anionic and cationic trypsins were located at exposed regions of the proteins.

The role of the carboxylate ion in models of acyl‐chymotrypsin

AbstractTwo exquisite models of the acyl‐chymotrypsin intermediate, one designed by Bruice and another by Bender, were studied to elucidate the role of the carboxylate ion in the catalytic triad of serine proteases. However, opposing conclusions were drawn from these studies. Computational chemistry analysis of these models indicates that the carboxylate ion plays an insignificant role in the former model because of the correct orientation and the distance of the imidazole group without the carboxylate ion. However, in the latter model, the carboxylate ion serves to orient and place the imidazole group at the correct position. This analysis suggests that an important role of the carboxylate ion in serine proteases is to position the imidazole group to be an effective general base catalyst. © 1995 by John Wiley & Sons, Inc.

Low-Barrier Hydrogen Bonding in Molecular Complexes Analogous to Histidine and Aspartate in the Catalytic Triad of Serine Proteases

We present spectroscopic evidence for the presence of low-barrier hydrogen bonds (LBHBs) in molecular complexes composed of carboxylic acids and 1-methylimidazole (1-MeIm) dissolved in aprotic organic solvents. A plot of the values of the low-field proton NMR chemical shifts versus the aqueous pKa of the carboxylic acid exhibits a positive slope for pKa values below 2.1 and a negative slope for higher pKa values. The chemical shifts for protons near the maximum in this plot are 18 ppm, similar to that of 18.3 ppm for His57-Asp102 in the protonated catalytic triad of chymotrypsin. The chemical shifts for the proton bonded to C2 of 1-MeIm in these complexes also vary with the pKa of the carboxylic acid and reveal a gradual change from neutral, hydrogen-bonded 1-MeIm in complexes of weaker acids to hydrogen-bonded 1-methylimidazolium ion in complexes of stronger acids. The midpoint chemical shift for the C2 proton corresponds to a carboxylic aqueous pKa of about 2.1. FTIR spectra of the 1-MeIm-carboxylic acid complexes in CHCl3 indicate that hydrogen bonding is strong and that the complexes are of three types: (a) neutral complexes with the weaker acids (pKa > or = 2.2) in which the antisymmetric carbonyl stretching frequencies are lowered relative to the free acids and the ethyl esters of the same acids; (b) ionic complexes of stronger acids (pKa < or = 2.1) in which the carbonyl stretching frequencies are slightly lower than those for the tetrabutylammonium salts of the same acids; (c) ionic complexes of the same acids (pKa < or = 2.1) coexisting with type b, in which the carbonyl stretching frequencies are intermediate between those for the tetrabutylammonium salts (bond order 1.5) and those of the same acids or their esters (bond order 2.0). The latter complexes appear to incorporate a low-barrier hydrogen bond and are presented as models for the protonated triad of chymotrypsin and other serine proteases. These enzymes have been postulated to utilize a low-barrier hydrogen bond between His57 and Asp102 to facilitate the abstraction of the beta-OH proton from Ser195 in the course of catalysis [Frey, P.A., Whitt, S.A., & Tobin, J.B. (1994) Science (Washington, D.C.)264,1927-1930].

Mechanistically different catalytic antibodies obtained from immunization with a single transition-state analog.

The variable-region peptide sequence and steady-state kinetic behavior are compared for a family of catalytic antibodies that arose from the same immune response to a transition-state analog. The crystal structure of the most catalytically active member of the family (17E8) has been solved to 2.5 A resolution and shows that the antibody active site contains a SerH99-HisH35 (H = heavy chain) catalytic dyad analogous to the Ser-His-Asp catalytic triad of serine proteases. The variable-region peptide sequence of the next most active antibody (29G11) differs from that of 17E8 by nine heavy-chain point mutations, and results from computer modeling suggest that the three-dimensional structure of 29G11 is similar to that of 17E8. In addition, 29G11 is an efficient catalytic antibody; it possesses 26% of the hydrolytic activity of 17E8. There is one active-site mutation in 29G11 compared to 17E8; position 99 of the heavy chain of 29G11 contains a glycine residue in place of the nucleophilic serine at this position in 17E8. Consistent with this mutation, results from pH-rate studies and hydroxylamine partitioning experiments indicate that in contrast to the catalytic mechanism of 17E8, the mechanism of 29G11-catalyzed esterolysis does not feature nucleophilic catalysis.

A new model for the agonistic binding site on the histamine H 2-receptor: The catalytic triad in serine proteases as a model for the binding site of

The historical model for the agonistic binding site on the histamine H 2-receptor is based on a postulated activation mechanism: it has been suggested that the histamine monocation binds to the histamine H 2-receptor via the formation of three hydrogen bonds. The cationic ammonium group in the side chain and the —NH— group in the π-position of the imidazole act as proton donors, whereas the N— atom in the π-position of the imidazole acts as a proton acceptor. Participation of the ammonium group in H-bonding with a presumed negative charge on the receptor leads to a decrease in positive charge, which is thought to induce a tautomeric change in the imidazole ring system from N τ-H to N π-H. A consequence of this tautomeric shift is the donation of a proton from the receptor to the agonist on one side, while on the other side a proton is donated from the agonist to the receptor. The proposed tautomeric shift has been suggested to trigger the H 2-stimulating effect. However, this model for the constitution of the agonistic binding site and the accessory activation mechanism cannot explain the weak histamine H 2-activity of β-histine and the activity of several other recently synthesized H 2-agonists. Based on a thorough literature study and with the aid of molecular electrostatic potentials (MEPs) we demonstrate that the sulphur atom present in histamine H 2-agonists as dimaprit and 2-amino-5-(2-aminoethyl)thiazole does not function as a proton acceptor, which implicitly means that a tautomeric shift is not a prerequisite for H 2-stimulation. As a consequence, the model for the agonistic binding site is adjusted, resulting in a strong resemblance to the nature and orientation of the amino acids constituting the catalytic triad in serine proteases. Within this concept, the N π-H tautomer of histamine is the biologically active form, in contrast with the existing model in which the N τ-H tautomer is the active form.

A Graph-theoretic Approach to the Identification of Three-dimensional Patterns of Amino Acid Side-chains in Protein Structures

This paper discusses the use of graph-theoretic methods for the representation and searching of three-dimensional patterns of side-chains in protein structures. The position a side-chain is represented by pseudo-atoms, and the relative positions of pairs of side-chains by the distances between them. This description of the geometry can be represented by a labelled graph in which the nodes and the edges of the graph represent the pseudo-atoms and the sets of inter-pseudo-atomic distances, respectively. Given such a representation, a protein can be searched for the presence of a user-defined query pattern of side-chains by means of a subgraph-isomorphism algorithm which is implemented in the program ASSAM. Experiments with one such algorithm, that due to Ullmann, show that it provides both an effective and a highly efficient way of searching for patterns of side-chains. The method is illustrated by searches for the serine protease catalytic triad, for residues involved in the catalytic activity of staphyloccocal nuclease, and for the zinc-binding side-chains of thermolysin. The catalytic triad pattern search revealed the existence of a second Asp-His-Ser-triad-like arrangement of residues in trypsinogen and chymotrypsinogen, in addition to the catalytic residues. In addition the program can be used to search for hypothetical patterns, as is shown for a pattern of three tryptophan side-chains. These searches demonstrate that the search algorithm can successfully retrieve the great majority of the expected proteins, as well as other, previously unreported proteins that contain the pattern of interest.

Crystal Structure of a Catalytic Antibody with a Serine Protease Active Site

The three-dimensional structure of an unusually active hydrolytic antibody with a phosphonate transition state analog (hapten) bound to the active site has been solved to 2.5 A resolution. The antibody (17E8) catalyzes the hydrolysis of norleucine and methionine phenyl esters and is selective for amino acid esters that have the natural alpha-carbon L configuration. A plot of the pH-dependence of the antibody-catalyzed reaction is bell-shaped with an activity maximum at pH 9.5; experiments on mechanism lend support to the formation of a covalent acyl-antibody intermediate. The structural and kinetic data are complementary and support a hydrolytic mechanism for the antibody that is remarkably similar to that of the serine proteases. The antibody active site contains a Ser-His dyad structure proximal to the phosphorous atom of the bound hapten that resembles two of the three components of the Ser-His-Asp catalytic triad of serine proteases. The antibody active site also contains a Lys residue to stabilize oxyanion formation, and a hydrophobic binding pocket for specific substrate recognition of norleucine and methionine side chains. The structure identifies active site residues that mediate catalysis and suggests specific mutations that may improve the catalytic efficiency of the antibody. This high resolution structure of a catalytic antibody-hapten complex shows that antibodies can converge on active site structures that have arisen through natural enzyme evolution.