Identification of catalytic and substrate-binding site residues in Bacillus cereus ATCC7064 oligo-1,6-glucosidase.

Three active site residues (Asp199, Glu255, Asp329) and two substrate-binding site residues (His103, His328) of oligo-1,6-glucosidase (EC 3.2.1.10) from Bacillus cereus ATCC7064 were identified by site-directed mutagenesis. These residues were deduced from the X-ray crystallographic analysis and the comparison of the primary structure of the oligo-1,6-glucosidase with those of Saccharomyces carlsbergensis alpha-glucosidase, Aspergillus oryzae alpha-amylase and pig pancreatic alpha-amylase which act on alpha-1,4-glucosidic linkages. The distances between these putative residues of B. cereus oligo-1,6-glucosidase calculated from the X-ray analysis data closely resemble those of A. oryzae alpha-amylase and pig pancreatic alpha-amylase. A single mutation of Asp199-->Asn, Glu255-->Gln, or Asp329-->Asn resulted in drastic reduction in activity, confirming that three residues are crucial for the reaction process of alpha-1,6-glucosidic bond cleavage. Thus, it is identified that the basic mechanism of oligo-1,6-glucosidase for the hydrolysis of alpha-1,6-glucosidic linkage is essentially the same as those of other amylolytic enzymes belonging to Family 13 (alpha-amylase family). On the other hand, mutations of histidine residues His103 and His328 resulted in pronounced dissimilarity in catalytic function. The mutation His328-->Asn caused the essential loss in activity, while the mutation His103-->Asn yielded a mutant enzyme that retained 59% of the k0/Km of that for the wild-type enzyme. Since mutants of other alpha-amylases acting on alpha-1,4-glucosidic bond linkage lost most of their activity by the site-directed mutagenesis at their equivalent residues to His103 and His328, the retaining of activity by His103-->Asn mutation in B. cereus oligo-1,6-glucosidase revealed the distinguished role of His103 for the hydrolysis of alpha-1,6-glucosidic bond linkage.     

Fluorescence technique for comparative studies of substrate-binding subsites in serine proteinases. Application to subtilisins.

A fluorescence technique for comparative studies of substrate-binding subsites in serine proteinases is described. It consists of: selective labelling of the corresponding subsites with a fluorescent group by using N alpha-dansyl(5-dimethylaminonaphthalene-1-sulphonyl)ated peptide chloromethanes containing different numbers of amino acid residues, and probing the immediate environment of the subsites by quenching experiments using ionic and neutral quenchers. Intramolecular distances between the subsites and particular chromophores can be also determined. The technique is of general applicability to all serine proteinases. The above mentioned approach was applied to two proteinases: subtilisin Novo and mesentericopeptidase. It was concluded that the substrate-binding site of mesentericopeptidase is considerably more polar than that of subtilisin Novo. Intramolecular distances between the labelled subsites and tryptophan residues in the two proteinases were determined.     

Prediction of substrate-binding site and elucidation of catalytic residue of a phytase from Bacillus sp

The present study primarily deals with the identification of substrate-binding site and elucidation of catalytic residue of the phytase from Bacillus sp. (Genbank Accession No. EF536824) employing molecular modeling and site-directed mutagenesis. Homology-based modeling of the Bacillus phytase revealed β-propeller structure with twelve active-site aminoacid residues, viz., D75, R77, Y78, H138, Q140, D189, D190, E191, Y238, Y239, N346 and R348. Docking of substrate Ins(1,2,3,4,5,6)hexakisphosphate with the phytase model disclosed interaction of Y78 residue with the sixth position phosphate, while D75 and R77 residues revealed hydrogen bonding with the fifth position phosphate of the phytate. Analysis of hydrolysis products of phytate indicated the sequential removal of alternate phosphates, resulting in the formation of final product Ins triphosphate. Mutant phytases Y78A/F, derived from site-directed mutagenesis, exhibited complete loss of enzyme activity despite substrate binding, thereby suggesting the intrinsic role of Y78 residue in the catalytic activity. The Bacillus mutant phytases can be used to generate enzyme crystals complexed with phytate and lower Ins phosphates for indepth analysis of substrate binding and catalytic activity of the enzyme.     

Site-directed mutagenesis in Bacillus stearothermophilus fructose-6-phosphate 1-kinase. Mutation at the substrate-binding site affects allosteric behavior

Arg252 of fructose-6-phosphate 1-kinase (PFK) from Bacillus stearothermophilus has been proposed to be involved in the binding of the substrate Fru-6-P. We demonstrate here that mutation of this residue to alanine converts the enzyme to a form with characteristics similar to those of its allosterically tight form. The mutant enzyme exhibits a high affinity for its inhibitor phosphoenolpyruvate (a 68-fold difference compared to wild type) and a dramatically decreased Fru-6-P affinity (1500-fold increase in Km). It is more sensitive to inhibition by high ATP concentrations than the wild type, and this inhibition is relieved by ADP, GDP, or higher Fru-6-P concentrations. In contrast, mutation of Arg252 to lysine increases the affinity of the enzyme for P-enolpyruvate by only 2-fold and increases its Km for Fru-6-P by only 50-fold. Sigmoidal kinetics with respect to Fru-6-P in the presence of P-enolpyruvate were observed with Hill numbers of 2.2, 2.4, and 1.7 for wild-type B. stearothermophilus PFK and the Arg252 to lysine and to alanine mutations, respectively. Unlike fructose-6-phosphate 1-kinase from Escherichia coli, in the absence of P-enolpyruvate, B. stearothermophilus PFK exhibits a hyperbolic profile with respect to Fru-6-P concentration. B. stearothermophilus PFK is sensitive to inhibition by high ATP concentrations and competitively inhibited by GDP or ADP. Our data indicate that Arg252 of B. stearothermophilus PFK plays a major role in both Fru-6-P binding and allosteric interaction between the subunits. However, this residue does not seem to participate directly in the catalytic process.     

Crystal structure of the high-alkaline serine protease PB92 from Bacillus alcalophilus.

The crystal structure of a serine protease from the alkalophilic strain Bacillus alcalophilus PB92 has been determined by X-ray diffraction at 1.75 A resolution. The structure has been solved by molecular replacement using the atomic model of subtilisin Carlsberg. The model of the PB92 protease has been refined to an R-factor of 14.0% and contains 1882 protein atoms, two calcium ions and 188 water molecules. The overall folding of the polypeptide chain closely resembles that of the subtilisins. Furthermore, almost all of the secondary structure elements found in subtilisin Carlsberg are also present in the PB92 protease. The major differences between the two structures are located around the deletion regions (residues 37 and 158-161 in subtilisin Carlsberg) and in two loops which are known to be the most variable parts of subtilisin structures. Flexibility of one of these loops (residues 126-130 in the PB92 protease) is believed to account for the induced-fit mechanism of substrate binding.     

Structure and function of the engineered multicopper oxidase CueO from Escherichia coli--deletion of the methionine-rich helical region covering the substrate-binding site

CueO is a multicopper oxidase (MCO) that is involved in the homeostasis of Cu in Escherichia coli and is the sole cuprous oxidase to have ever been found. Differing from other MCOs, the substrate-binding site of CueO is deeply buried under a methionine-rich helical region including alpha-helices 5, 6, and 7 that interfere with the access of organic substrates. We deleted the region Pro357-His406 and replaced it with a Gly-Gly linker. The crystal structures of a truncated mutant in the presence and in the absence of excess Cu(II) indicated that the scaffold of the CueO molecule and metal-binding sites were reserved in comparison with those of CueO. In addition, the high thermostability of the protein molecule and its spectroscopic and magnetic properties due to four Cu centers were also conserved after truncation. As for functions, the cuprous oxidase activity of the mutant was reduced to ca 10% that of recombinant CueO owing to the decrease in the affinity of the labile Cu site for Cu(I) ions, although activities for laccase substrates such as 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), p-phenylenediamine, and 2,6-dimethoxyphenol increased due to changes in the access of these organic substrates to the type I Cu site. The present engineering of CueO indicates that the methionine-rich alpha-helices function as a barrier to the access of bulky organic substrates, which provides CueO with specificity as a cuprous oxidase.     

Photoreactivity of histidyl residues in subtilisins Novo and DY. Photooxidation of subtilisins

Subtilisins Novo and DY were photoinactivated in the presence of methylene blue according to first order kinetics. The competitive inhibitor N alpha-benzoyl-L-arginine protected significantly against inactivation. Under the conditions employed in this study a selective photooxidation of the active site histidine 64 was achieved. Rate constants of 0.32 X 10(-2), s-1 and 0.35 X 10(-2), s-1, were calculated for the Novo enzyme and subtilisin DY, respectively. Apparent pKa values of the catalytically important imidazole group of 7.0 +/- 0.1 (s. Novo) and 7.1 +/- 0.1 (s. DY) were directly determined. The histidyl residues in the two proteases, except the active site histidine, which is the first target of photooxidation, are "buried" in the interior of the protein globule. Conformational studies suggested that the photoreactive histidine is not involved in the stabilization of the protein conformation.     

Transmembrane reorientation of the substrate-binding site in vesicular acetylcholine transporter

Active transport of acetylcholine (ACh) by vesicular ACh transporter (VAChT) is driven by a proton-motive force established by V-ATPase. A published microscopic kinetics model predicts the ACh-binding site is primarily oriented toward the outside for nontransporting VAChT and toward the inside for transporting VAChT. The allosteric ligand [(3)H]vesamicol cannot bind when the ACh-binding site is outwardly oriented and occupied by ACh, but it can bind when the ACh site is inwardly oriented. The kinetics model was tested in the paper reported here using rat VAChT expressed in PC12(A1237) cells. Equilibrium titrations of [(3)H]vesamicol binding and ACh competition show that ATP blocks competition between vesamicol and ACh in over one-half of the VAChT. NaCl did not mimic ACh chloride, and bafilomycin A(1) and FCCP completely blocked the ATP effect, which shows that it is mediated by a proton-motive force. The data are consistent with reorientation of over one-half of the ACh-binding sites from the outside to the inside of vesicles upon activation of transport. The observations support the proposed microscopic kinetics model, and they should be useful in characterizing effects of mutations on the VAChT transport cycle.     

Intracellular serine protease of Bacillus subtilis: sequence homology with extracellular subtilisins.

Intracellular serine protease was isolated from stationary-grown Bacillus subtilis A-50 cells and purified to homogeneity. The molecular weight of the enzyme is 31,000 +/- 1,000, with an isoelectric point of 4.3. Its amino acid composition is characteristically enriched in glutamic acid content, differing from that of extra-cellular subtilisins. The enzyme is completely inhibited with phenylmethylsulfonyl fluoride and ethylenediaminetetraacetic acid. Intracellular protease possesses negligible activity towards bovine serum albumin and hemoglobin, but has 5- to 20-fold higher specific activity against p-nitroanilides of benzyloxycarbonyl tripeptides than subtilisin BPN'. Esterolytic activity of the enzyme is also higher than that of subtilisin BPN'. The enzyme is sequence homologous with secretory subtilisins throughout 50 determined NH2-terminal residues, indicating the presence of duplicated structural genes for serine proteases in the B. subtilis genome. The occurrence of two homologous genes in the cell might accelerate the evolution of serine protease not only by the loosening of selective constrainst, but also by creation of sequence variants by means of intragenic recombination. Three molecular forms of intracellular protease were found, two of them with NH2-terminal glutamic acid and one minor form, three residues longer, with asparagine as NH2 terminus. These data indicate the possible presence of an enzyme precursor proteolytically modified during cell growth.     

Surface-simulation synthesis of the substrate-binding site of an enzyme. Demonstration with trypsin.

From the X-ray co-ordinates of bovine trypsin and its complexes with substrate analogues (benzamidine) and with soya-bean trypsin inhibitor, a peptide (TP) was designed and synthesized by surface-simulation synthesis, a concept previously introduced by this laboratory, to mimic the binding site of trypsin. Also, a control peptide (CTP) was synthesized that contained all the amino acids present in the TP peptide, except that their order was randomized. The radioiodinated TP peptide bound specifically to adsorbents of benzamidine, whereas the control CTP peptide exhibited no binding activity. Conjugates to succinyl (3-carboxypropionyl)-lysozyme of the TP peptide, control CTP peptide and other unrelated peptides were examined by a radiometric binding assay for the ability to bind soya-bean trypsin inhibitor and human alpha 1-antitrypsin. Conjugates of the TP peptide exhibited considerable binding activity to adsorbents of soya-bean trypsin inhibitor or alpha 1-antitrypsin. None of the other peptide conjugates possessed any binding activity. Action of the active-site-directed reagents phenylmethanesulphonyl fluoride and di-isopropyl phosphorofluoridate on free TP and CTP peptides resulted in the modification of a serine residue in the TP peptide whereas the CTP peptide remained unaltered. The TP peptide, either in the free form or as a conjugate on succinyl-lysozyme, had no enzymic activity on protein substrates or on tosylarginine methyl ester. These findings indicated that the binding activity of an enzyme was well mimicked by the surface-stimulation peptide but that reproduction of the catalytic activity was not obtained.     

Proton magnetic resonance studies of the states of ionization of histidines in native and modified subtilisins

A technique was developed to exchange the backbone -N-H protons in D2O in the native subtilisins Carlsberg and BPN (Novo) that resulted in clearly resolved proton resonances in the aromatic region of the nuclear magnetic resonance spectrum. pH titration curves for four of the five histidine C2-H resonances in subtilisin Carlsberg and five of the six in subtilisin BPN between 7.5 and 8.8 ppm downfield from 4,4-dimethyl-4-silapentane-1-sulfonic acid sodium salt provided microscopic pKa's between 6.3 and 7.2 for both sources of the enzyme at ambient (approximately 22 degrees C) probe temperature. A resonance that titrated with a pKapp of 7.35 +/- 0.05 was observed in the 1H spectra only of the diisopropylphosphoryl derivatives of the subtilisins from both sources. The 31P NMR pH titration of the same preparations under identical conditions of solvent (D2O) and temperature gave a pKapp = 7.40 +/- 0.05 of the single titratable resonance. Both observations must pertain to His-64 at the active center. A resonance smaller than the others and titrating with a pKapp of 7.2 could also be observed in the native enzymes. This resonance was assigned to the catalytic center histidine since its pK corresponded to that derived from kinetic studies. No major perturbations in the chemical shifts or the pK's derived from the pH dependence of the observed resonances were apparent in the presence of saturating concentrations of the two putative transition-state analogues phenylboronic acid and bis [3,5-(trifluoromethyl)phenyl]boronic acid and in monoisopropylphosphorylsubtilisin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anion transport in red blood cells and arginine-specific reagents. Interaction between the substrate-binding site and the binding site of arginine-specific reagents

Phenylglyoxal is found to be a potent inhibitor of sulfate equilibrium exchange across the red blood cell membrane at both pH 7.4 and 8.0. The inactivation exhibits pseudo-first-order kinetics with a reaction order close to one at both pH 7.4 and 8. The rate constant of inactivation at 37 degrees C was found to be 0.12 min-1 at pH 7.4 and 0.19 min-1 at pH 8.0. Saturation kinetics are observed if the pseudo-first order rate constant of inhibition is measured as a function of phenylglyoxal concentration. Sulfate ions as well as chloride ions markedly decrease the rate of inactivation by phenylglyoxal at pH 7.4, suggesting that the modification occurs at or near to the binding site for chloride and sulfate. The decrease of the rate of inactivation produced at pH 8.0 by chloride ions is much higher than that produced by sulfate ions. Kinetic analysis of the protection experiments showed that the loaded transport site is unable to react with phenylglyoxal. From the data it is concluded that the modified amino acid(s) residues, presumably arginine, is (are) important for the binding of the substrate anion.     

Brain pyridoxal kinase: photoaffinity labeling of the substrate-binding site

4-Benzoylbenzoic acid inhibits pyridoxal kinase activity competitively with respect to pyridoxal. The Ki was determined to be 5 x 10(-5) M. Binding studies showed that 4-benzoylbenzoic acid bound to pyridoxal kinase at a 1:1 molar ratio and with a dissociation constant (Kd) of 5.9 x 10(-5) M. Photoirradiation of pyridoxal kinase in the presence of a 10-fold excess of 4-benzoylbenzoic acid at pH 6.5 resulted in an irreversible loss of enzymatic activity; this photoinactivation was prevented by the presence of pyridoxal. Amino acid analysis revealed that 1 tyrosine residue/subunit was modified during photoinactivation. The presence of a tyrosine residue at the active site of pyridoxal kinase was confirmed by reaction with tetranitromethane. In the presence of 1 x 10(-4) M tetranitromethane, a complete loss of the kinase activity was observed after incubation at 25 degrees C for 8 min, with modification of a total of 3 tyrosine residues. The second-order rate constant (K2) of the reaction between the tyrosine residues and tetranitromethane was determined to be 53.3 s-1 M-1.     

Functional definition of the tobacco protoporphyrinogen IX oxidase substrate-binding site

PPO (protoporphyrinogen IX oxidase) catalyses the flavin-dependent six-electron oxidation of protogen (protoporphyrinogen IX) to form proto (protoporphyrin IX), a crucial step in haem and chlorophyll biosynthesis. The apparent K(m) value for wild-type tobacco PPO2 (mitochondrial PPO) was 1.17 muM, with a V(max) of 4.27 muM.min(-1).mg(-1) and a catalytic activity k(cat) of 6.0 s(-1). Amino acid residues that appear important for substrate binding in a crystal structure-based model of the substrate docked in the active site were interrogated by site-directed mutagenesis. PPO2 variant F392H did not reveal detectable enzyme activity indicating an important role of Phe(392) in substrate ring A stacking. Mutations of Leu(356), Leu(372) and Arg(98) increased k(cat) values up to 100-fold, indicating that the native residues are not essential for establishing an orientation of the substrate conductive to catalysis. Increased K(m) values of these PPO2 variants from 2- to 100-fold suggest that these residues are involved in, but not essential to, substrate binding via rings B and C. Moreover, one prominent structural constellation of human PPO causing the disease variegate porphyria (N67W/S374D) was successfully transferred into the tobacco PPO2 background. Therefore tobacco PPO2 represents a useful model system for the understanding of the structure-function relationship underlying detrimental human enzyme defects.     

Photoaffinity labeling identifies the substrate-binding site of mammalian squalene epoxidase

Squalene epoxidase (SE) catalyzes the conversion of squalene to (3S)-2,3-oxidosqualene. Photolabeling and site-directed mutagenesis were performed on recombinant rat SE (rrSE) in order to identify the location of the substrate-binding site and the roles of key residues in catalysis. Truncated 50-kDa rrSE was purified and photoaffinity labeled by competitive SE inhibitor (Ki=18.4 microM), [(3)H]TNSA-Dza. An 8-kDa CNBr/BNPS-skatole peptide was purified and the first 24 amino acids were sequenced by Edman degradation. The sequence PASFLPPSSVNKRGVLLLGDAYNL corresponded to residues 388-411 of the full-length rat SE. Three nucleophilic residues (Lys-399, Arg-400, and Asp-407) were labeled by [(3)H]TNSA-Dza. Triple mutants were prepared in which bulky groups were used to replace the labeled charged residues. Purified mutant enzymes showed lower enzymatic activity and reduced photoaffinity labeling by [(3)H]TNSA-Dza. This constitutes the first evidence as to the identity of the substrate-binding site of SE.     

Altered active site flexibility and a structural metal-binding site in eukaryotic dUTPase: kinetic characterization, folding, and crystallographic studies of the homotrimeric Drosophila enzyme

dUTPase is responsible for preventive DNA repair via exclusion of uracil. Developmental regulation of the Drosophila enzyme is suggested to be involved in thymine-less apoptosis. Here we show that in addition to conserved dUTPase sequence motifs, the gene of Drosophila enzyme codes for a unique Ala-Pro-rich segment. Kinetic and structural analyses of the recombinant protein and a truncation mutant show that the Ala-Pro segment is flexible and has no regulatory role in vitro. The homotrimer enzyme unfolds reversibly as a trimeric entity with a melting temperature of 54 degrees C, 23 degrees C lower than Escherichia coli dUTPase. In contrast to the bacterial enzyme, Mg(2+) binding modulates conformation of fly dUTPase, as identified by spectroscopy and by increment in melting temperature. A single well folded, but inactive, homotrimeric core domain is generated through three distinct steps of limited trypsinolysis. In fly, but not in bacterial dUTPase, binding of the product dUMP induces protection against proteolysis at the tryptic site reflecting formation of the catalytically competent closed conformer. Crystallographic analysis argues for the presence of a stable monomer of Drosophila dUTPase in crystal phase. The significant differences between prototypes of eukaryotic and prokaryotic dUTPases with respect to conformational flexibility of the active site, substrate specificity, metal ion binding, and oligomerization in the crystal phase are consistent with alteration of the catalytic mechanism and hydropathy of subunit interfaces.     

Investigation of myosin substrate-binding site using phosphorylating analogs of the substrate

Affinity modification of HMM has been performed, using mixed anhydrides of AMP, epsilon AMP, ADP or IMP and sterically hindered aromatic carbonic acids. The affinity labelling site of HMM was demonstrated to be highly specific towards the adenosine fragment of the affinity analog. The number of phosphate groups and the hydrophobicity of the aromatic acid substitutents did not influence the mode of the analogs interaction with HMM. The data obtained confirms our previous suggestion that the nucleotide analogs in question modify the substrate binding site which is other than the active site of the enzyme.     

Mutation in the substrate-binding site of aminopeptidase B confers new enzymatic properties

Aminopeptidase B (Ap-B) catalyzes the cleavage of arginine and lysine residues at the N-terminus of various peptide substrates. In vivo, it participates notably in the miniglucagon and cholecystokinin 8 processing, but the complete range of physiological functions of Ap-B remains to be discovered. Ap-B is a member of the M1 family of Zn²⁺-metallopeptidases that are characterized by two highly conserved motives, GXMEN (potential substrate binding site) and HEXXHX¹⁸E (Zn²⁺-binding site). In this study, mutagenesis and molecular modelling were used to investigate the enzymatic mechanism of Ap-B. Nineteen rat Ap-B mutants of the G₂₉₈XM₃₀₀E₃₀₁N₃₀₂ motif and one mutant of the HEIS₃₂₈HX¹⁸E motif were expressed in Escherichia coli. All mutations except G₂₉₈P, G₂₉₈S, and S₃₂₈A abolished the aminopeptidase activity. The S₃₂₈A mutant mimics the sequence of bovine Ap-B Zn²⁺-binding site, which differs from those of other mammalian Ap-B. This mutant conserved a canonical Ap-B activity. G₂₉₈S and G₂₉₈P mutants exhibit new enzymatic properties such as changes in their profile of inhibition and their sensitivity to Cl⁻ anions. Moreover, the G₂₉₈P mutant exhibits new substrate specificity. A structural analysis using circular dichroism, fluorescence spectroscopy, molecular modelling and dynamics was performed to investigate the role that residue G₂₉₈ plays in the catalytic mechanism of Ap-B. Our results show that G₂₉₈ is essential to Ap-B activity and participates to the substrate specificity of the enzyme.