Enzyme-catalysed conjugations of glutathione with unsaturated compounds

1. Rat-liver supernatant catalyses the reaction of diethyl maleate with glutathione. 2. Evidence is presented that the enzyme involved is different from the known glutathione-conjugating enzymes, glutathione S-alkyltransferase, S-aryltransferase and S-epoxidetransferase. 3. Rat-liver supernatant catalyses the reaction of a number of other αβ-unsaturated compounds, including aldehydes, ketones, lactones, nitriles and nitro compounds, with glutathione: separate enzymes may be responsible for these reactions.     

Functional expression of trypsin from Streptomyces griseus by Pichia pastoris

In the present study, the genes encoding trypsinogen and active trypsin from Streptomyces griseus were both cloned and expressed in the methylotrophic yeast Pichia pastoris with the α-factor secretion signal under the control of the alcohol oxidase promoter. The mature trypsin was successfully accumulated extracellularly in soluble form with a maximum amidase activity of 6.6?U?ml^?1 (batch cultivation with flask cultivation) or 14.4?U?ml^?1 (fed-batch cultivation with a 3-l fermentor). In contrast, the recombinant trypsinogen formed inclusion bodies and no activity was detected. Replacement of the trypsin propeptide Ala-Pro-Asn-Pro confirmed that its physiological function was as a repressor of activity. More importantly, our results proved that the propeptide inhibited the activity of trypsinogen after its successful folding.     

Occurrence and properties of a thermostable protease secreted by sea-urchin embryos

1. A trypsinogen-activating enzyme is distinct from the chorion-dissolving enzyme of the sea-urchin blastula, but is secreted with it. 2. This ;trypsinogenase' does not attack N-alpha-toluene-p-sulphonylarginine methyl ester, and is not inhibited by concentrations of di-isopropyl phosphorofluoridate or 7-amino-1-chloro-3-toluene-p-sulphonamidoheptan-2-one that are 98% effective against trypsin. It requires a small amount of Ca(2+) for activity, but is inhibited by more, from as little as 1mm. 3. Enzymic activity is associated with a wide range of particle weights, some apparently as high as 15x10(6). The mean particle weight can be decreased by sonication. 4. Fresh trypsinogenase in the higher range of particle weights appears to be remarkably stable to heating, e.g. retaining full activity after boiling for 15min.     

Fetuin as a trypsin inhibitor

Fetuin per se is a moderately strong trypsin inhibitor of the temporary type and retains its antitryptic activity upon desialicization. It forms a reversible 1:1 complex with β-trypsin and is functionally homogeneous in this respect. Complex formation is an entropy-driven reaction evidently due to release of structured water associated with the individual proteins. It is concluded that the contact area in the complex probably includes the active site of trypsin and that dissociation of the complex is rapid.     

FAD and FMN flavoproteins participate in the sodium-transport respiratory chain NADH:quinone reductase of a marine bacterium,Vibrio alginolyticus

The sodium-transport respiratory chain NADH:quinone reductase of a marine bacterium, Vibrio alginolyti-cus, is composed of three protein subunits, α,β and γ. The β-subunit contains FAD as a prosthetic group and corresponds to NADH dehydrogenase, which catalyses the reduction of ubiquinone to ubisemiquinone. In addition to β, subunits α. and γ are essential for the quinone reductase, which catalyses the reduction of ubiquinone to ubiquinol. The α-subunit contains FMN and the reaction catalysed by subunit α is related to the coupling site of the sodium pump in the quinone reductase.     

Sucrose synthase catalyses a readily reversible reaction in vivo in developing potato tubers and other plant tissues

Experiments were carried out to investigate whether sucrose synthase (Susy) catalyses a readily reversible reaction in vivo in potato (Solanum tuberosum L.) tubers, Ricinus communis L. cotyledons, and heterotrophic Chenopodium rubrum L. cell-suspension cultures. (i) The contents of sucrose, fructose, UDP and UDP-glucose were measured and the mass-action ratio compared with the theoretical equilibrium constant. In all three tissues the values were similar. (ii) Evidence for rapid turnover of label in the sucrose pool was obtained in pulse-chase experiments with potato discs and with intact tubers attached to the plant. The unidirectional rates of sucrose synthesis and degradation were considerably higher than the net flux through the sucrose pool in the tubers. (iii) Labelling of the glucosyl and fructosyl moieties of sucrose from [¹⁴C]glucose in the presence of unlabelled fructose provided evidence that Susy contributes to the movement of label into sucrose. Methods for estimating the contribution of sucrose-phosphate synthase and Susy are presented and it is shown that their relative contribution varies. For example, the contribution of Susy is high in developing tubers and is negligible in harvested tubers which contain low Susy activity. (iv) The absolute values of the forward (v⁺¹) and backward (v^?1) reaction direction of Susy are calculated from the kinetic labelling data. The estimated values of v⁺¹ and v^?1 are comparable, and much higher than the net flux through the sucrose pool. (v) The estimated concentrations of the substrates and products of Susy in tubers are comparable to the published K _m values for potato-tuber Susy. (vi) It is concluded that Susy catalyses a readily reversible reaction in vivo and the relevance of this conclusion is discussed with respect to the regulation of sucrose breakdown and the role of Susy in phloem unloading.     

4-Hydroxyphenylacetate decarboxylase activating enzyme catalyses a classical S-adenosylmethionine reductive cleavage reaction

4-Hydroxyphenylacetate decarboxylase (4Hpad) is an Fe/S cluster containing glycyl radical enzyme (GRE), which catalyses the last step of tyrosine fermentation in clostridia, generating the bacteriostatic p-cresol. The respective activating enzyme (4Hpad-AE) displays two cysteine-rich motifs in addition to the classical S-adenosylmethionine (SAM) binding cluster (RS cluster) motif. These additional motifs are also present in other glycyl radical activating enzymes (GR-AE) and it has been postulated that these orthologues may use an alternative SAM homolytic cleavage mechanism, generating a putative 3-amino-3-carboxypropyl radical and 5′-deoxy-5′-(methylthio)adenosine but not a 5′-deoxyadenosyl radical and methionine. 4Hpad-AE produced from a codon-optimized synthetic gene binds a maximum of two [4Fe–4S]^2+/+ clusters as revealed by EPR and Mössbauer spectroscopy. The enzyme only catalyses the turnover of SAM under reducing conditions, and the reaction products were identified as 5′-deoxyadenosine (quenched form of 5′-deoxyadenosyl radical) and methionine. We demonstrate that the 5′-deoxyadenosyl radical is the activating agent for 4Hpad through p-cresol formation and correlation between the production of 5′-deoxyadenosine and the generation of glycyl radical in 4Hpad. Therefore, we conclude that 4Hpad-AE catalyses a classical SAM-dependent glycyl radical formation as reported for GR-AE without auxiliary clusters. Our observation casts doubt on the suggestion that GR-AE containing auxiliary clusters catalyse the alternative cleavage reaction detected for glycerol dehydratase activating enzyme.     

Preparation and characterization of mixed-mode magnetic adsorbent with p-amino-benzamidine ligand: Operated in a magnetically stabilized fluidized bed reactor for purification of trypsin from bovine pancreas

The magnetic beads were synthesized using glycidylmethacrylate (GMA) and methylmethacrylate (MMA) monomers. A multimodal ligand (i.e., p-amino-benzamidine) was covalently immobilized onto magnetic beads after glutaraldehyde activation, and consequently used for purification of the trypsin from bovine pancreas. The p-amino-benzamidine ligand immobilized magnetic beads were characterized by FTIR, VSM, SEM, and analytical methods. Trypsin adsorption experiments were investigated under different experimental conditions (i.e., medium pH, initial trypsin concentration, temperature, and ionic strength) in a batch system. Maximum trypsin adsorption capacity was found to be 75.9 ± 2.6 mg/g beads. Adsorbed trypsin was eluted by using (0.1 M acetate buffer, pH 3.0) with a 97% recovery. The purification factor of trypsin from crude pancreas extract was 8.7 folds. The purity of the eluted trypsin from p-amino-benzamidine functionalized magnetic beads was determined as 86% by HPLC. The method developed in this report was successfully applied for purification of the trypsin from crude pancreas extract in a magnetically stabilized fluidized bed reactor.     

Enzyme substrate and inhibitor interactions.

An enzyme is designed to bind most tightly to a substrate when it is in the transition state of the reaction which the enzyme catalyses. The consequent reduction of the activation energy of the reaction constitutes the catalytic mechanism. The energetic contributions of different features of the interaction can only be crudely assessed, but they are dominated by entropically driven effects. The binding site of trypsin orients the substrate so that the reacting groups are correctly placed for reaction to occur. Apart from two side chains which take part in chemical steps of the reaction, the enzyme behaves almost as a rigid body. The full binding interactions are only developed when the substrate is in an intermediate stage of the reaction. The tightly bound complexes of trypsin with protein trypsin inhibitors have proved amenable to structural analysis. Enzyme inhibitor interactions, which account for almost 80 kJ mol-1 of interaction energy, are known fairly accurately. The similarity of the two known trypsin inhibitor structures, close to the primary binding site, indicates a high specificity, even for this simple interaction. In cases where no large conformational changes occur the specificity of an enzyme should be predictable from accurate knowledge of its tertiary structure.     

Mechanism of biotin carboxylase inhibition by ethyl 4-[[2-chloro-5-(phenylcarbamoyl)phenyl]sulphonylamino]benzoate

The rise of antibacterial-resistant bacteria is a major problem in the United States of America and around the world. Millions of patients are infected with antimicrobial resistant bacteria each year. Novel antibacterial agents are needed to combat the growing and present crisis. Acetyl-CoA carboxylase (ACC), the multi-subunit complex which catalyses the first committed step in fatty acid synthesis, is a validated target for antibacterial agents. However, there are at present, no commercially available antibiotics that target ACC. Ethyl 4-[[2-chloro-5-(phenylcarbamoyl)phenyl]sulfonylamino]benzoate (SABA1) is a compound that has been shown to have antibacterial properties against Pseudomonas aeruginosa and Escherichia coli. SABA1 inhibits biotin carboxylase (BC), the enzyme that catalyses the first half reaction of ACC. SABA1 inhibits BC via an atypical mechanism. It binds in the biotin binding site in the presence of ADP. SABA1 represents a potentially new class of antibiotics that can be used to combat the antibacterial resistance crisis.     

Sequential assembly of photosynthetic units in Rhodobacter sphaeroides as revealed by fast repetition rate analysis of variable bacteriochlorophyll a fluorescence

The development of functional photosynthetic units in Rhodobacter sphaeroides was followed by near infra-red fast repetition rate (IRFRR) fluorescence measurements that were correlated to absorption spectroscopy, electron microscopy and pigment analyses. To induce the formation of intracytoplasmic membranes (ICM) (greening), cells grown aerobically both in batch culture and in a carbon-limited chemostat were transferred to semiaerobic conditions. In both aerobic cultures, a low level of photosynthetic complexes was observed, which were composed of the reaction center and the LH1 core antenna. Interestingly, in the batch cultures the reaction centers were essentially inactive in forward electron transfer and exhibited low photochemical yields F_V/F_M, whereas the chemostat culture displayed functional reaction centers with a rather rapid (1-2 ms) electron transfer turnover, as well as a high F_V/F_M of ∼0.8. In both cases, the transfer to semiaerobiosis resulted in rapid induction of bacteriochlorophyll a synthesis that was reflected by both an increase in the number of LH1-reaction center and peripheral LH2 antenna complexes. These studies establish that photosynthetic units are assembled in a sequential manner, where the appearance of the LH1-reaction center cores is followed by the activation of functional electron transfer, and finally by the accumulation of the LH2 complexes.     

Modifying the determinants of α-ketoacid substrate selectivity in mycobacterium tuberculosis α-isopropylmalate synthase

α-Isopropylmalate synthase (IPMS) catalyses the reaction between α-ketoisovalerate and acetyl coenzyme A (AcCoA) in the first step of leucine biosynthesis. IPMS is closely related to homocitrate synthase, which catalyses the reaction between AcCoA and the unbranched α-ketoacid α-ketoglutarate. Analysis of these enzymes suggests that several differently conserved key residues are responsible for the different substrate selectivity. These residues were systematically substituted in the Mycobacterium tuberculosis IPMS, resulting in changes in substrate specificity. A variant of IPMS was constructed with a preference for the unbranched α-ketoacids α-ketobutyrate and pyruvate over the natural branched substrate α-ketoisovalerate.     

Effect of the Medium Dielectric Strength on the Activity of Alpha Chymotrypsin

The rates of hydrolysis of TrEE, TEE, and ATEE¹ by α-chymotrypsin were determined in media of variable dielectric strength. Many substances which modify the dielectric constant of the medium, exert additional specific effects on the reaction rate, noticeable at more or less elevated concentrations. Notwithstanding, it is possible to differentiate the dielectric and specific effects by comparing the rates in solvents of distinct nature at relatively low concentrations. Thus, the effect of varying the dielectric strength could be studied within wider ranges (ΔD = 20 with TrEE and ca. 28 with ATEE) than in the previous study of trypsin (ΔD = 12). The dielectric effect on α-chymotrypsin is the opposite of that observed with trypsin. In both cases there is a linear relationship between the logarithm of the rate of hydrolysis and the reciprocal of the dielectric constant. The slope is negative with α-chymotrypsin and positive with trypsin. According to expressions relating the dielectric constant to the rate in non-enzymatic reactions, the behavior of α-chymotrypsin is like that of a negative ion, while trypsin behaves as a positive ion. The enzyme activity appears to depend upon the arrangement of charges in the enzyme and substrate molecules, rather than on the presence of certain atomic groupings in the substrate.     

Initiation of the degradation of the soybean kunitz and bowman-birk trypsin inhibitors by a cysteine protease

Protease K1 activity initiates the degradation of the Kunitz soybean trypsin inhibitor (KSTI) during germination and early seedling growth. This enzyme was purified nearly 1300-fold from the cotyledons of 4-day-old soybean (Glycine max [L.] Merrill) seedlings. Protease K1 is a cysteine protease with a molecular weight of approximately 29,000. It cleaves the native form of KSTI, Ti^a, to Ti^a_m, the same modified form observed in vivo. In addition to attacking KSTI, protease K1 is also active toward the major Bowman-Birk soybean trypsin inhibitor, as well as the α, α′, and β subunits of soybean β-conglycinin. The properties and temporal variation of protease K1 during germination indicate that it is responsible for initiating the degradation of both KSTI and Bowman-Birk soybean trypsin inhibitor in the soybean cotyledon.     

Transphosphatidylation reaction of phosphatidylcholine to 4-methoxyphenol in water-immiscible organic solvents with immobilized phospholipase D

Immobilized phospholipase D (PLD) from Streptomyces sp. catalyzed the transfer reaction of the dipalmitoylphosphatidyl residue from 1,2-dipalmitoyl-3-sn-phosphatidylcholine (DPPC) to an aromatic hydroxy group on 4-methoxyphenol in water-immiscible organic solvents, to afford 1,2-dipalmitoyl-3-sn-phosphatidyl-4-methoxyphenol (DPP-PMP) with a 45% yield, accompanied by a trace amount of 1,2-dipalmitoyl-3-sn-phosphatidic acid sodium salt (DPPA-Na). The formation of DPP-PMP was affected by organic solvents used in the reaction. Benzene, toluene, and methylene chloride gave DPP-PMP with moderate yields but use of diethyl ether resulted in a low yield of DPP-PMP. In both ethyl acetate and water-miscible organic solvents, the transfer reaction did not take place. Immobilization of PLD was carried out by adding a 1 % volume of PLD solution to a suspension of a cation-exchange resin (Amberlite IRC-50, 5% w/v) in benzene with stirring and sonication. In a repeated batch reaction for DPP-PMP synthesis with immobilized PLD, after ten batch cycles the enzyme retained 74% of its initial activity.     

In vivo and in vitro effects of wheat germ agglutinin and Bowman-Birk soybean trypsin inhibitor,two potential transgene products,on midgut esterase and protease activities from Apis mellifera

Wheat germ agglutinin (WGA) and Bowman-Birk soybean trypsin inhibitor represent potential transgene products for inducing pest resistance in plants. The effects of these molecules were studied on midgut esterase and protease activities from Apis mellifera L., a major insect pollinator. Trypsin inhibitor and WGA did not exhibit an acute toxicity in A. mellifera. In vivo, trypsin inhibitor caused a decrease in the amount of trypsin activity and did not have a significant effect on esterase activity. In vitro, trypsin inhibitor inhibited about 80% of non-specific protease activity and 100% of trypsin activity. In vivo, WGA at high concentration in food (1 mg/ml) elicited a large decrease in trypsin activity and did not have a significant effect on esterase activity. In vitro, WGA did not have any significant effect on trypsin and non-specific protease activities but slightly activated esterase activity.     

Catechol O-methyltransferases in pampas grass: differentiation of m- and p-methylating activities

Partially purified catechol O-methyltransferase from pampas grass (Cortaderia selloana) catalyses the methylations of substrates at both their meta and para positions. This capability was shown, by heat treatments, to arise from a less stable m-O-methyl-transferring activity and a more stable p-O-methyltransferring activity, tested against protocatechuic acid. When acting upon caffeic acid, the preparation catalyses a reaction of solely m-O-methyltransfer (in contrast to the mixed methylation of this substrate exhibited by rat liver catechol O-methyltransferase). A small degree of m-O-methylation of monophenolic substrates also occurs.     

Electrostatic effects play a central role in cold adaptation of trypsin.

Organisms that live in constantly cold environments have to adapt their metabolism to low temperatures, but mechanisms of enzymatic adaptation to cold environments are not fully understood. Cold active trypsin catalyses reactions more efficiently and binds ligands more strongly in comparison to warm active trypsin. We have addressed this issue by means of comparative free energy calculations studying the binding of positively charged ligands to two trypsin homologues. Stronger inhibition of the cold active trypsin by benzamidine and positively charged P1-variants of BPTI is caused by rather subtle electrostatic effects. The different affinity of benzamidine originates solely from long range interactions, while the increased binding of P1-Lys and -Arg variants of BPTI is attributed to both long and short range effects that are enhanced in the cold active trypsin compared to the warm active counterpart. Electrostatic interactions thus provide an efficient strategy for cold adaptation of trypsin.     

Characterisation of a PS II reaction centre isolated from the chloroplasts of Pisum sativum

A photosystem II reaction centre has been isolated from peas and found to consist of D1, D2 polypeptides and the apoproteins of cytochrome b-559, being similar to that reported for spinach by Nanba and Satoh [(1987) Proc. Natl. Acad. Sci. USA 84, 109–112]. The complex binds chlorophyll a, pheophytin and the haem of cytochrome b-559 in an approximate ratio of 4:2:1 and also contains about one molecule of β-carotene. It binds no plastoquinone-9 or manganese but does contain at least one non-haem iron. In addition to a light-induced signal due to Pheo⁻ seen under reducing conditions, a light-induced P680⁺ signal is seen when the reaction centre is incubated with silicomolybdate. In the presence of diphenylcarbazide, the P680⁺ signal is partially inhibited and net electron flow to silicomolybdate occurs. This net electron flow is insensitive to o-phenanthroline, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea and 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene but is inhibited by proteolysis with trypsin and by other treatments. Fluorescence, from the complex, peaks at 682 nm at room temperature and at 685 nm at 77 K. This emission is significantly quenched when either the P680⁺Pheo or P680Pheo⁻ states are established indicating that the fluorescence emanates from the back reaction between P680⁺ and Pheo⁻.