The carboxyl terminus of coffee bean alpha-galactosidase is critical for enzyme activity

The role of the carboxyl (C)-terminal region of coffee bean alpha-galactosidase (alpha-GAL) has been studied by expressing C-terminal deletion mutants in the methylotrophic yeast strain Pichia pastoris. A previous study of human alpha-galactosidase determined that enzyme activity increased when up to 10 amino acid residues were deleted. Deleting 11 residues reduced activity, and deleting 12 residues abolished activity. In our studies, alpha-GAL activity is reduced when one or two amino acids are deleted, as is enzyme secretion directed by P. pastoris signal sequences. The pH profile is similar to that of the wild-type enzyme. Deleting 3 or more residues from the C-terminal end results in a complete loss of both enzyme secretion and activity. The C-terminus of alpha-GAL seems to play an important role in overall enzyme conformation and may directly affect the proper conformation of the active site.     

Glycosidases of Ehrlich ascites tumor cells and ascitic fluid--purification and substrate specificity of alpha-N-acetylgalactosaminidase and alpha-galactosidase: comparison with coffee bean alpha-galactosidase

Ehrlich ascites tumor cells and ascitic fluid were assayed for glycosidase activity. alpha-Galactosidase and beta-galactosidase, alpha- and beta-mannosidase, alpha-N-acetylgalactosaminidase, and beta-N-acetylglucosaminidase activities were detected using p-nitrophenyl glycosides as substrates. alpha-Galactosidase and alpha-N-acetylgalactosaminidase were isolated from Ehrlich ascites tumor cells on epsilon-aminocaproylgalactosylamine-Sepharose. alpha-Galactosidase was purified 160,000-fold and was free of other glycosidase activities. alpha-N-Acetylgalactosaminidase was also purified 160,000-fold but exhibited a weak alpha-galactosidase activity which appears to be inherent in this enzyme. Substrate specificity of the alpha-galactosidase was investigated with 12 substrates and compared with that of the corresponding coffee bean enzyme. The pH optimum of the Ehrlich cell alpha-galactosidase centered near 4.5, irrespective of substrate, whereas the pH optimum of the coffee bean enzyme for PNP-alpha-Gal was 6.0, which is 1.5 pH units higher than that for other substrates of the coffee bean enzyme. The reverse was found for alpha-N-acetylgalactosaminidase: the pH optimum for the hydrolysis of PNP-alpha-GalNAc was 3.6, lower than the pH 4.5 required for the hydrolysis of GalNAc alpha 1,3Gal. Coffee bean alpha-galactosidase showed a relatively broad substrate specificity, suggesting that it is suited for cleaving many kinds of terminal alpha-galactosyl linkages. On the other hand, the substrate specificity of Ehrlich alpha-galactosidase appears to be quite narrow. This enzyme was highly active toward the terminal alpha-galactosyl linkages of Ehrlich glycoproteins and laminin, both of which possess Gal alpha 1, 3Gal beta 1,4GlcNAc beta-trisaccharide sequences. The alpha-N-acetylgalactosaminidase was found to be active toward the blood group type A disaccharide, and trisaccharide, and glycoproteins with type A-active carbohydrate chains.     

The synthesis, testing and use of 5-fluoro-alpha-D-galactosyl fluoride to trap an intermediate on green coffee bean alpha-galactosidase and identify the catalytic nucleophile

5-Fluoro-alpha-D-galactopyranosyl fluoride was synthesized and its interaction with the active site of an alpha-galactosidase from green coffee bean (Coffea arabica), a retaining glycosidase, characterized kinetically and structurally. The compound behaves as an apparently tight binding (Ki = 600 nM) competitive inhibitor, achieving this high affinity through reaction as a slow substrate that accumulates a high steady-state concentration of the glycosyl-enzyme intermediate, as evidenced by ESiMS. Proteolysis of the trapped enzyme coupled with HPLC/MS analysis allowed the localization of a labeled peptide that was subsequently sequenced. Comparison of this sequence information to that of other members of the same glycosidase family revealed the active site nucleophile to be Asp145 within the sequence LKYDNCNNN. The importance of this residue to catalysis has been confirmed by mutagenesis studies.     

Mechanistic insight into the catalytic inhibition by nitroxides of tyrosine oxidation and nitration

BackgroundNitroxide antioxidants (RNO^•) protect from injuries associated with oxidative stress. Tyrosine residues in proteins are major targets for oxidizing species giving rise to irreversible cross-linking and protein nitration, but the mechanisms underlying the protective activity of RNO^• on these processes are not sufficiently clear.MethodsTyrosine oxidation by the oxoammonium cation (RN⁺=O) was studied by following the kinetics of RNO^• formation using EPR spectroscopy. Tyrosine oxidation and nitration were investigated using the peroxidase/H₂O₂ system without and with nitrite. The inhibitory effect of RNO^• on these processes was studied by following the kinetics of the evolved O₂ and accumulation of tyrosine oxidation and nitration products.ResultsTyrosine ion is readily oxidized by RN⁺=O, and the equilibrium constant of this reaction depends on RNO^• structure and reduction potential. RNO^• catalytically inhibits tyrosine oxidation and nitration since it scavenges both tyrosyl and ^•NO₂ radicals while recycling through RN⁺=O reduction by H₂O₂, tyrosine and nitrite. The inhibitory effect of nitroxide on tyrosine oxidation and nitration increases as its reduction potential decreases where the 6-membered ring nitroxides are better catalysts than the 5-membered ones.ConclusionsNitroxides catalytically inhibit tyrosine oxidation and nitration. The proposed reaction mechanism adequately fits the results explaining the dependence of the nitroxide inhibitory effect on its reduction potential and on the concentrations of the reducing species present in the system.General significanceNitroxides protect against both oxidative and nitrative damage. The proposed reaction mechanism further emphasizes the role of the reducing environment to the efficacy of these catalysts.     

New insight into the catalytic properties of bile salt hydrolase

Bile salt hydrolase (BSH), the enzyme deconjugating bile potentially plays an important role in reduction of blood cholesterol level. BSH enzymes from various sources differ in characteristics, substrates preference and specific catalytic activity. In this study, two BSH enzymes (BSH1 and BSH2) from Lactobacillus salivarius were heterologously expressed and purified. Both of them were characterized as homotetramer according to their molecular weight from size exclusion chromatograph. BSH1 showed a broad pH optimum over the range from 5.5 to 7.0, while a narrower range of pH optimum from 5.5 to 6.0 for BSH2 was detected. The enzymatic kinetics of the purified BSH1 and BSH2 have demonstrated BSH enzymes from bacteria were allosteric enzymes, and have also revealed their striking differences in positive cooperativity, catalytic efficiency and substrate preference for the first time. In contrast to the enzymatic reactions of BSH in the absence of dithiothreitol, the kinetics curves of BSH1 and BSH2 were similar to hyperbolic forms of Michaelis–Menten kinetics in the presence of dithiothreitol.     

Forces, bond lengths, and reactivity: fundamental insight into the mechanism of enzyme catalysis.

Comparison of spectroscopic, kinetic, and thermodynamic data for a series of functioning acylserine proteases suggests that the observed variation in deacylation rates can be accounted for by changes in the properties of the acyl-enzyme's ground state. The acyl-enzyme's catalytically crucial acyl carbonyl group is probed by resonance Raman spectroscopy. Its spectral frequency is used to gauge both the carbonyl bond length and the strength of hydrogen bonding (originating from groups making up the oxyanion hole) to the carbonyl oxygen atom. As the deacylation rate increases 16,300-fold through the series, a shift in carbonyl frequency, vC = O, of -54 cm-1 corresponds to a carbonyl bond length increase of 0.025 A. The decrease in vC = O is also consistent with an increase in hydrogen bond donor enthalpy of -27 kJ mol-1. Interestingly, this value resembles closely the decrease in activation energy for deacylation through the series, 24 kJ mol-1, demonstrating that the hydrogen bonds to the carbonyl oxygen atom can provide sufficient energy to account for the observed rate accelerations.     

Altitude and coffee production systems influence extent of infestation and bean damage by the coffee berry borer

The coffee berry borer (CBB), Hypothenemus hampei Ferrari (Coleoptera: Curculionidae: Scolytinae), is one of the major insect pests of coffee worldwide. The present study was designed to assess the level of infestation of coffee berries at different developmental stages across different altitudes and coffee management systems. The experiment was carried out at three locations in southwestern Ethiopia under two coffee management systems and four coffee berry development stages with three replications. Results of the study showed significantly highest proportion of damaged berries (37.5%), number of holes per berry (10.88) and number of adult CBB per berry (7.55) on dried leftover berries at low-altitude study sites. On the other hand, the lowest mean percent damaged berries, number of holes per berry and number of adults were recorded at mid- and high-altitude study sites. The study also showed that, CBB caused significantly highest damage in plantation coffee management system than garden coffee. Results of this study highlight proper harvesting at red ripe stage in order to minimise incidence of CBB. It is also important to design integrated management strategies to mitigate CBB damage especially in lowland plantation coffee production systems.     

Alpha-galactosyl fluoride in transfer reactions mediated by the green coffee beans alpha-galactosidase in ice

We show that the yields in saccharide synthesis by tranglycosylation with alpha-galactosidase from green coffee beans can be greatly enhanced when working in ice. Thus, methyl alpha-D-galactopyranosyl-(1-->3)-alpha-D-galactopyranoside (3a) produced by reaction of alpha-D-galactopyranosyl fluoride 1 with methyl alpha-D-galactopyranoside (2) is obtained with 51% yield in ice while only 29% is synthesized at 37 degrees C. This result, already previously found by others with proteases and by us with a beta-galactosidase appears to be a general property of hydrolases.     

Crystal structure of glycerophosphodiester phosphodiesterase (GDPD) from Thermoanaerobacter tengcongensis, a metal ion-dependent enzyme: insight into the catalytic mechanism

Glycerophosphodiester phosphodiesterase (GDPD; EC 3.1.4.46) catalyzes the hydrolysis of a glycerophosphodiester to an alcohol and glycerol 3-phosphate in glycerol metabolism. It has an important role in the synthesis of a variety of products that participate in many biochemical pathways. We report the crystal structure of the Thermoanaerobacter tengcongensis GDPD (ttGDPD) at 1.91 A resolution, with a calcium ion and glycerol as a substrate mimic coordinated at this calcium ion (PDB entry 2pz0). The ttGDPD dimer with an intermolecular disulfide bridge and two hydrogen bonds is considered as the potential functional unit. We used site-directed mutagenesis to characterize ttGDPD as a metal ion-dependent enzyme, identified a cluster of residues involved in substrate binding and the catalytic reaction, and we propose a possible general acid-base catalytic mechanism for ttGDPD. Superposing the active site with the homologous structure GDPD from Agrobacterium tumefaciens (PDB entry 1zcc), which binds a sulfate ion in the active site, the sulfate ion can represent the phosphate moiety of the substrate, simulating the binding mode of the true substrate of GDPD.     

The crystal structure of bacillus cereus phosphonoacetaldehyde hydrolase: insight into catalysis of phosphorus bond cleavage and catalytic diversification within the HAD enzyme superfamily

Phosphonoacetaldehyde hydrolase (phosphonatase) catalyzes the hydrolysis of phosphonoacetaldehyde to acetaldehyde and phosphate using Mg(II) as cofactor. The reaction proceeds via a novel bicovalent catalytic mechanism in which an active-site nucleophile abstracts the phosphoryl group from the Schiff-base intermediate formed from Lys53 and phosphonoacetaldehyde. In this study, the X-ray crystal structure of the Bacillus cereus phosphonatase homodimer complexed with the phosphate (product) analogue tungstate (K(i) = 50 microM) and the Mg(II) cofactor was determined to 3.0 A resolution with an R(cryst) = 0.248 and R(free) = 0.284. Each monomer is made up of an alpha/beta core domain consisting of a centrally located six-stranded parallel beta-sheet surrounded by six alpha-helices. Two flexible, solvated linkers connect to a small cap domain (residues 21-99) that consists of an antiparallel, five-helix bundle. The subunit-subunit interface, formed by the symmetrical packing of the two alpha8 helices from the respective core domains, is stabilized through the hydrophobic effect derived from the desolvation of paired Met171, Trp164, Tyr162, Tyr167, and Tyr176 side chains. The active site is located at the domain-domain interface of each subunit. The Schiff base forming Lys53 is positioned on the cap domain while tungstate and Mg(II) are bound to the core domain. Mg(II) ligands include two oxygens of the tungstate ligand, one oxygen of the carboxylates of Asp12 and Asp186, the backbone carbonyl oxygen of Ala14, and a water that forms a hydrogen bond with the carboxylate of Asp190 and Thr187. The guanidinium group of Arg160 binds tungstate and the proposed nucleophile Asp12, which is suitably positioned for in-line attack at the tungsten atom. The side chains of the core domain residue Tyr128 and the cap domain residues Cys22 and Lys53 are located nearby. The identity of Asp12 as the active-site nucleophile was further evidenced by the observed removal of catalytic activity resulting from Asp12Ala substitution. The similarity of backbone folds observed in phosphonatase and the 2-haloacid dehalogenase of the HAD enzyme superfamily indicated common ancestry. Superposition of the two structures revealed a conserved active-site scaffold having distinct catalytic stations. Analysis of the usage of polar amino acid residues at these stations by the dehalogenases, phosphonatases, phosphatases, and phosphomutases of the HAD superfamily suggests possible ways in which the active site of an ancient enzyme ancestor might have been diversified for catalysis of C-X, P-C, and P-O bond cleavage reactions.     

Solid state NMR: new tools for insight into enzyme function

NMR has had considerable impact in enzymology, probing evidence for ionization states, conformational 'strain', compressed interactions, electronically unusual species, and conformational dynamics of enzymes. Solid-state NMR is becoming increasingly important in studying enzymes because of a number of recent tools for analysis of proteins by SSNMR, and because of the growing ability to isolate the species of interest for analysis. Here, we review recent studies of a Michaelis complex, of the dynamic functioning of membrane-associated enzymes, and initial studies of several enzymes with redox-active and paramagnetic centers.     

Structural insight into the cooperativity between catalytic and noncatalytic sites of F1-ATPase

F1-ATPase, the catalytic sector of Fo-F1 ATPases-ATPsynthases, displays an apparent negative cooperativity for ATP hydrolysis at high ATP concentrations which involves noncatalytic and catalytic nucleotide binding sites. The molecular mechanism of such cooperativity is currently unknown. To get further insights, we have investigated the structural consequences of the single mutation of two residues: Q173L in the alpha-subunit and Q170Y in the beta-subunit of the F1-ATPase of the yeast Schizosaccharomyces pombe. These residues are localized in or near the Walker-A motifs of each subunit and their mutation produces an opposite effect on the negative cooperativity. The betaQ170 residue (M167 in beef heart) is located close to the binding site for the phosphate-Mg moiety of the nucleotide. Its replacement by tyrosine converts this site into a close state with increased affinity for the bound nucleotide and leads to an increase of negative cooperativity. In contrast, the alphaQ173L mutation (Q172 in beef heart) abolishes negative cooperativity due to the loss of two H-bonds: one stabilizing the nucleotide bound to the noncatalytic site and the other linking alphaQ173 to the adjacent betaT354, localized at the alpha(DP)-beta(TP) interface. The properties of these mutants suggest that negative cooperativity occurs through interactions between neighbor alpha- and beta-subunits. Indeed, in the beef heart enzyme, (i) the alpha(DP)-beta(TP) interface is stabilized by a vicinal alphaR171-betaD352 salt bridge (ii) betaD352 and betaT354 belong to a short peptidic stretch close to betaY345, the aromatic group of which interacts with the adenine moiety of the nucleotide bound to the catalytic site. We therefore propose that the betaY345-betaT354 stretch (beef heart numbering) constitutes a short link that drives structural modifications from a noncatalytic site to the neighbor catalytic site in which, as a result, the affinity for ADP is modulated.     

The role of Tyr248 probed by mutant bovine carboxypeptidase A: insight into the catalytic mechanism of carboxypeptidase A

We have investigated the function of Tyr248 using bovine wild-type CPA and its Y248F and Y248A mutants to find that the K(M) values were increased by 4.5-11-fold and the k(cat) values were reduced by 4.5-10.7-fold by the replacement of Tyr248 with Phe for the hydrolysis of hippuryl-L-Phe (HPA) and N-[3-(2-furyl)acryloyl]-Phe-Phe (FAPP), respectively. In the case of O-(trans-p-chlorocinnamoyl)-L-beta-phenyllactate (ClCPL), an ester substrate, the K(M) value was increased by 2.5-fold, and the k(cat) was reduced by 20-fold. The replacement of Tyr248 with Ala decreased the k(cat) values by about 18- and 237-fold for HPA and ClCPL, respectively, demonstrating that the aromatic ring of Tyr248 plays a critical role in the enzymic reaction. The increases of the K(M) values were only 6- and 5-fold for HPA and ClCPL, respectively. Thus, the present study indicates clearly that Tyr248 plays an important role not only in the binding of substrate but also in the enzymic hydrolysis. The kinetic results may be rationalized by the proposition that the phenolic hydroxyl of Tyr248 forms a hydrogen bond with the zinc-bound water molecule, causing further activation of the water molecule by reducing its pK(a) value. The pH dependency study of k(cat) values and the solvent isotope effects also support the proposition. A unified catalytic mechanism is proposed that can account for the different kinetic behavior observed in the CPA-catalyzed hydrolysis of peptide and ester substrates.     

Modeling studies of potato nucleoside triphosphate diphosphohydrolase NTPDase1: an insight into the catalytic mechanism

Nucleoside triphosphate diphosphohydrolase--NTPDase1 (apyrase, EC 3.6.1.5) was modeled based on sequence homology. The single polypeptide chain of apyrase is folded into two domains. The putative catalytic site with the apyrase conserved regions (ACR 1-5) is located between these two domains. Modeling confirmed that apyrase belongs to the actin superfamily of proteins. The amino acids interacting with the nucleoside triphosphate substrate and probably involved in the catalyzed hydrolysis were identified. The proposed two-step catalytic mechanism of hydrolysis involves Thr127 and Thr55 as potential nucleophilic factors responsible for the cleavage of the Pgamma and Pbeta anhydride bonds, respectively. Their action seems to be assisted by Glu170 and Glu78 residues, respectively. The presence of two nucleophiles in the active site of apyrase explains the differences in the hydrolytic activity between apyrases and other enzymes belonging to the NTPDase family.