Internal thermodynamics of enzymes determined by equilibrium quench: values of Kint for enolase and creatine kinase

The equilibrium constant (Kint) for the enzyme-bound substrate and product of a one substrate/one product enzyme (enolase) and for those of a two substrate/two product enzyme (creatine kinase) have been determined. The values of Kint were determined by the rapid quenching of equilibrium mixtures of enzyme and radiolabeled substrate and product, under conditions where all of the marker substrate and product are bound. The scope and limitations of this method are discussed. Values of Kint have been collected from the literature, and it is shown that these data are consistent with the theory for kinetically optimized enzymes that is developed in the preceding paper.     

Process for the aerobic conversion of poultry manure into high-protein feedstuff

A two-stage aerobic continuous process is suggested for the conversion of poultry waste into a single-cell protein (SCP) product. The technical feasibility of the process is examined in this paper. Using bench-scale apparatus the approximate growth kinetics have been discovered. Possible modes of product recovery have been examined. The product consists primarily of a strain of Pseudomonas fluorescens which has a lysine-rich (～9.3%) amino acid profile that could make this product an attractive feed ingredient for poultry diets.     

Identification of bacteriophage T4D gene products 26 and 51 as baseplate hub structural components

Products of two bacteriophage T4D genes, 26 and 51, both known to be essential for the formation of the central hub of the phage tail baseplate, have been partially characterized chemically, and their biological role has been examined. The gene 26 product was found to be a protein with a molecular size of 41,000 daltons and the gene 51 product a protein of 16,500 daltons. The earlier proposal (L. M. Kozloff and J. Zorzopulos, J. Virol. 40:635-644), from observations of a 40,000-dalton protein in labeled hubs, that the gene 26 product is a structural component of the baseplate, has been confirmed. The gene 51 product, not yet detected in phage particles, appears from indirect evidence also to be a structural component of the baseplate hub. These current conclusions about the gene 26 and 51 products are based on properties of T4 mutant particles containing altered gene 26 or 51 products and include (i) changes in heat lability, (ii) changes in adsorption rates, and (iii) changes in plating efficiencies on different hosts, and with the results of previous isotope incorporation experiments indicate that T4 particles contain three copies of the gene 26 product and possibly one or at most two copies of the gene 51 product. Properties of these mutant particles indicate that the gene 26 product, together with the other hub components such as the gene 28 product, plays a critical role in phage DNA injection into the host cell, whereas the 51 product seems essential in initiating baseplate hub assembly.     

Function of dopachrome oxidoreductase and metal ions in dopachrome conversion in the eumelanin pathway

The conversion of dopachrome (DC) in the eumelanin pathway has been analyzed to determine the specific product and the role of enzyme control. 5,6-Dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) were quantitated by HPLC with fluorescent detection, after DC incubation with heated and unheated preparations of B-16 melanoma derived dopachrome oxidoreductase (DCOR). The enzyme-catalyzed reaction produced DHICA as the major product, while DHI formed with the spontaneous reaction. It had originally been suggested that the major product of DC conversion was DHI, with DHICA being formed as a minor product of this conversion [Raper, H.S. (1927) Biochem. J. 21, 89-96]. Copper, nickel, and cobalt ions promoted conversion of DC, with nickel simulating DCOR activity. Removal of free ions from unheated DCOR did not alter DC conversion. We conclude that the major product of DC conversion is DHICA and that DCOR is responsible for this conversion.     

Cloning and sequencing of the beta-fructofuranosidase gene from Bacillus sp. V230.

The beta-fructofuranosidase gene (bff) from Bacillus sp. V230 has been cloned in Escherichia coli and its nucleotide sequence has been analyzed. The product of bff consists of a signal sequence of 32 amino acid (a.a.) residues for secretion and 455 a.a. residues of the extracellular beta-fructofuranosidase. The a.a. sequence of the bff product has similarities with those of the Bacillus subtilis levanscrase (63.7% identity), the Streptococcus mutans fructosyltransferase (33.7%), and the Zymomonas mobilis levanscrase and beta-fructofuranosidase (15%).     

Nonlinear kinetics of the xanthine oxidase reaction catalyzed by chicken liver xanthine dehydrogenase

The xanthine oxidase reaction catalyzed by chicken liver xanthine dehydrogenase has been shown to give nonlinear kinetics of the type which has been identified as substrate activation. When a very wide range of substrate (pteridine) concentrations were studied, it was found that a downward deflection in reciprocal plots (substrate activation) occurs in the high region and an upward deflection in the very low region. When product (isoxanthopterin) was included in reaction mixtures, the upward deflection was enhanced and shifted to higher substrate concentration ranges. In addition, reciprocal plots with a second substrate (oxygen) and a product (isoxanthopterin) were nonlinear.     

Catalytic mechanism of aromatic prenylation by NphB

NphB is an aromatic prenyltransferase that catalyzes the attachment of a 10-carbon geranyl group to aromatic substrates. Importantly, NphB exhibits a rich substrate selectivity and product regioselectivity. A systematic computational study has been conducted in order to address several question associated with NphB-catalyzed geranylation. The reaction mechanism of the prenylation step has been characterized as a S(N)1 type dissociative mechanism with a weakly stable carbocation intermediate. A novel π-chamber composed of Tyr121, Tyr216, and 1,6-DHN is found to be important in stabilizing the carbocation. The observed difference in the rates of product formation from 5- and 2-prenylation arises from the differing orientations of the aromatic substrate in the resting state. 4-Prenylation shares the same resting state with 5-prenylation, but the lower free energy barrier for carbocation formation makes the latter reaction more facile. The high free energy barrier associated with 7-prenylation is caused by the unfavorable orientation of 1,6-DHN in active site pocket, along with the difficulty of proton elimination after the prenylation step. A water-mediated proton transfer facilitates the loss of hydrogen at the prenylation site to form the final prenylated product. Interestingly, the same crystallographically observed water molecule has been found to be responsible for proton loss in all three experimentally identified products. After proton transfer, the relaxation of the final product from a sp(3) carbon center to a sp(2) center triggers a "spring-loaded" product release mechanism which pushes the final product out of the binding pocket toward the edge of the active site. The hydrogen bond interactions between the two hydroxyl groups of the aromatic product and the side chains of Ser214 and Tyr288 help to "steer" the movement of the product. In addition, mutagenesis studies identify these same two side chains as being responsible for the observed regioselectivity, particularly 2-prenylation. These observations provide valuable insights into NphB chemistry, offering an opportunity to better engineer the active site and to control the reactivity in order to obtain high yields of the desired product(s). Furthermore, the S(N)1 reaction mechanism observed for NphB differs from the prenylation reaction found in, for example, the farnesyltransferase, which proceeds via an S(N)2-like reaction pathway. The spring-loaded release mechanism highlighted herein also offers novel insights into how enzymes facilitate product release.     

A concentration-dependent localization of octopamine-sensitive adenylate cyclase activity in locust skeletal muscle

A concentration-dependent localization of octopamine-sensitive adenylate cyclase activity has been demonstrated in skeletal muscle of the locust, Schistocerca gregaria, using an histochemical technique. In the intermediate speed contracting muscle fibres from the fan region of the extensor-tibiae muscle of the locust hindleg, low concentrations of DL-octopamine (10(-8) M) induce reaction product preferentially in the sarcoplasmic reticular component of the dyads. At slightly higher concentrations (10(-7) and 10(-6) M) lower amounts of diffuse reaction product are also found in the non-dyad sarcoplasmic reticulum and at the sarcolemmal membrane, with occasional amounts of a less diffuse, punctuate product in the transverse tubule (T-tubule) component of the dyads. At higher concentrations (10(-5) and 10(-3) M) the predominant product is the dense, plaque-like accumulations of reaction product in the T-tubule component of the dyads. The results are discussed in terms of the likely physiological significance of the accumulation of reaction product in these different locations.     

Synthesis of phenyl-adducted cyclodextrin through the click reaction

A new derivative of β-cyclodextrin (CD) has been made incorporating the phenyl group through the use of click reaction. The resulting product suggests a self-association phenomenon through the formation of inclusion compound between the phenyl group and CD. The product has been characterized by ¹H and ¹³C NMR spectroscopy.     

Cloning, mapping and gene product identification of rhaT from Escherichia coli K12

Rhamnose utilization requires the function of a specific rhamnose transport system. Rhamnose transport mutants have been isolated and characterized. The structural gene, rhaT, encoding the rhamnose permease has been cloned from Escherichia coli. rhaT has been mapped in the rha locus (87.7 min) by analysis of cotransduction with glpK and other rha markers. The precise location of the gene has been determined by complementation analysis of rhamnose transport mutants transformed with recombinant plasmids containing different fragments of the cloned region. Gene order (counterclockwise) is established as glpK . . . rhaT-rhaR-rhaS-rhaB-rhaA-rhaD. The gene product has been identified by expression of rhaT in a T7 RNA polymerase/promoter system. This 23 kDa protein has been assigned to the rhaT product and has been shown to be located in the cell membrane.     

Isolation of a cyclitol antibiotic: 2,3,4,5-tetrahydroxycyclohexanemethanol

The isolation and structure determination of a new microbial product, (+)- (123/45)-2,3,4,5-tetrahydroxy-1-cyclohexanemethanol is described. This product was detected in the fermentation broth of a newly isolated actinomycete by its antibacterial activity. A novel isolation method was developed and crystalline product was obtained in good yield. The structure was determined by spectroscopic examination of the product and its acetyl and trimethylsilyl derivatives. The racemic form of this compound had already been synthezised by G. E. McCasland et. al., as analog of galactose.     

Oxygenated iron bleomycin. A short-lived intermediate in the reaction of ferrous bleomycin with O2.

The reaction of Fe(II) . bleomycin with O2 to yield Fe(III) . bleomycin has been resolved into two kinetic events by stopped-flow spectrophotometry. The first event is first order with respect to both bleomycin and O2 and may be regarded as a second order reaction (k = 6.1 x 10(3) M-1s-1 at 2 degrees C). The first product has no EPR spectrum. The optical spectrum resembles those of Fe(II) . bleomycin complexes with CO, NO, and ethyl isocyanide. We propose that the first product is an Fe(II) . bleomycin . O2 complex. The second kinetic event is first order with respect to the first accumulated product (k = 0.11 s-1 at 2 degrees C) and independent of oxygen concentration. The product of this reaction is indistinguishable from Fe(III) . bleomycin by optical and EPR spectroscopy.     

Drying of ethanol vapors by adsorption on corn meal

Experiments have been carried out on a laboratory-scale adsorber packed with 3.3 kg cornmeal. The effects of varying particle size, feed concentrations and flow rates, and regeneration conditions on the product quality and breakthrough times were investigated. It was found that 85% (w/w) vapor feed gave the best product, and product yields of up to 99.8% (w/w) alcohol were obtained at energy costs as low as 4.0 MJ kg(-1). It is suggested how this figure could be improved.     

Benzidine dihydrochloride as a chromogen for single- and double-label light and electron microscopic immunocytochemical studies

Although very sensitive chromogens have been adapted for localization of horseradish peroxidase in anterograde and retrograde tracing studies, they have not been successfully applied in immunocytochemical studies. This report describes a protocol which uses benzidine dihydrochloride (BDHC) as the chromogen for light (LM) and electron microscopic (EM) immunocytochemical studies. The protocol is comparable to that used for tetramethylbenzidine, except that the pH of the reaction is above 6.0. At the LM level, the BDHC reaction product is bluish-green and crystalline. Both the color and form of the product are readily distinguished from the reddish-brown DAB reaction product. LM double-labeling studies are therefore feasible. The use of BDHC also increases significantly the sensitivity of the immunoreaction. Higher fixative concentrations can be used, less detergent is necessary, and higher primary antibody dilutions are possible. By osmicating at 45 degrees C in an s-collidine buffer it is possible to preserve the soluble BDHC reaction product for EM analysis. Immunoreactive cells are particularly well labeled with this new protocol. The BDHC crystals are easily detected at the EM level and can be distinguished from flocculent DAB reaction product. This feature makes EM double-labeling studies possible.     

Peptide-based inhibitors of hepatitis C virus full-length NS3 (protease-helicase/NTPase): model compounds towards small molecule inhibitors

From L-alpha-aminobutyric acid (Abu) a set of electrophilic and non-electrophilic replacements for the P1 cysteine of substrate and product inhibitors of hepatitis C virus full-length NS3 (protease-helicase/NTPase) serine protease have been synthesised and coupled to a model pentapeptide furnishing a set of hexapeptide inhibitors. Promising inhibitory activities with K(i) values of 0.18 microM (11b, P1 electrophilic alpha,beta-unsaturated ketone), 0.46 microM (12e, P1 electrophilic alkyl ketone) and 0.98 microM (10e, P1 non-electrophilic alkenyl alcohol as diastereomeric mixture). The reference hexapeptide product inhibitor had a K(i) value of 1.54 microM (14, P1 Abu-OH). The electrophilic inhibitors exhibit increased potency as compared with the corresponding product inhibitor, and notably also the non-electrophilic P1 alkenyl alcohol 10e. This represents the first example of non-electrophilic inhibitors that are not P1 amides or product inhibitors.     

Chemometrics applications in biotech processes: assessing process comparability

A typical biotech process starts with the vial of the cell bank, ends with the final product and has anywhere from 15 to 30 unit operations in series. The total number of process variables (input and output parameters) and other variables (raw materials) can add up to several hundred variables. As the manufacturing process is widely accepted to have significant impact on the quality of the product, the regulatory agencies require an assessment of process comparability across different phases of manufacturing (Phase I vs. Phase II vs. Phase III vs. Commercial) as well as other key activities during product commercialization (process scale-up, technology transfer, and process improvement). However, assessing comparability for a process with such a large number of variables is nontrivial and often companies resort to qualitative comparisons. In this article, we present a quantitative approach for assessing process comparability via use of chemometrics. To our knowledge this is the first time that such an approach has been published for biotech processing. The approach has been applied to an industrial case study involving evaluation of two processes that are being used for commercial manufacturing of a major biosimilar product. It has been demonstrated that the proposed approach is able to successfully identify the unit operations in the two processes that are operating differently. We expect this approach, which can also be applied toward assessing product comparability, to be of great use to both the regulators and the industry which otherwise struggle to assess comparability.     

Biosynthesis of pulmonary glycoconjugates. Mechanism of glucosylation of protein and lipid acceptors

In sheep lung microsomes, we have shown that glucosyl-transferases catalize the transfer of glucose from UDP-glucose into four different acceptors. The glucosylated products obtained are as follows: - a glucosyl-phosphoryl-polyprenol (product A) extractable by chloroform/methanol (2:1 by volume). - a product B extractable by chloroform/methanol/water (10:10:3 by volume). The product B was a mixture of five glycolipids, one of them having a chromatographic behaviour similar to the behaviour of a tetrahexosylceramide (asia-lo-GM1). - a product C insoluble in water and organic solvents which has been demonstrated to be a glycoproteinic compound. The molecular weight of this product C was 160 000 as estimated by gel-filtration. The carbohydrate moiety is composed of small oligosaccharides which are found to be attached by O-glycosidic bond to the protein chain. This linkage is not a collagen-like bond.     

Kinetics of the hydrolysis of cellulose by beta-1,4-glucan cellobiohydrolase of Trichoderma viride

The cellulolytic enzyme beta-1,4-glucan cellobiohydrolase (CBH) has been isolated from the crude mixture of cellulase enzymes of Trichoderma viride by gel filtration and ion-exchange methods, and some aspects of its kinetic behaviour have been examined. Studies of the initial rates of the CBH-catalyzed production of cellobiose from fibrous alpha-cellulose show that (i) the dissociation constant for cellobiose competitive product inhibition of the reaction is Ki = (1.13 +/- 0.37) X 10(-3) M, (ii) the adsorption of CBH on fibrous alpha-cellulose and its subsequent reaction conform to kinetic equations developed in conjunction with the Langmuir adsorption isotherm, (iii) the rate-pH curve has a maximum at pH 5.2 and decreases at higher and lower pH values, exhibiting enzyme pK values of 3.8 and 6.5, and (iv) the energy of activation of the overall reaction between 5 and 60 degrees C is 5.3 +/- 0.3 kcal mol-1 at pH 5.2. Studies of the time course of the reaction over extended periods of time up to 40% hydrolysis of the cellulose show that (v) the data fit better to a competitive product inhibition model than to models of anticompetitive product inhibition or noncompetitive product inhibition.     

The polyA regions of hen oviduct RNA

Total RNA from hen oviduct has been hydrolysed with a mixture of T₁ and pancreatic ribonucleases. Poly(A) tracts in the digestion product have been isolated by binding to oligo(dT) cellulose. Of the four major ribonucleotides, the product has been shown to contain only adenylic acid. When separated on polyacrylamide gels, the poly(A) gave two peaks corresponding to average apparent lengths of 270-280 and 540-550 nucleotides.