Penicillin acylase catalyzed synthesis of penicillin-G from substrates anchored in cyclodextrins.

Penicillin acylase (EC 3.5.1.11) catalyses the condensation of phenylacetic acid (PAA) and 6-aminopenicillanic acid (6-APA) to form benzylpenicillin (BP). Both PAA and 6-APA were found to form host-guest complexes with beta-methylcyclodextrin (beta m-CD) and gamma-cyclodextrin (gamma-CD) respectively. The rate of the reaction catalyzed by the enzyme remained unaffected if one of the substrates used was in the cyclodextrin complexed form. However, in this case, the reaction lasted longer and yielded about 20 per cent more products compared to the condensation reaction involving only uncomplexed substrates. There was distinct increase in the rate of formation of the antibiotic, if both substrates used are in CD-complexed form.

Affinity properties of phosvitin: interaction of phosvitin with serine hydroxymethyl transferase.

The affinity of phosvitin with serine hydroxymethyl transferase (SHMT), an acidic multi-subunit protein, was evaluated by measurements of enzyme activity, sedimentation velocity, steady-state fluorescence, circular dichroism and kinetic thermal stability. While the presence of phosvitin had no effect on the SHMT activity, the sedimentation coefficient of SHMT increased from 8.7 S to 12.5 S suggesting the formation of a complex at a SHMT:phosvitin molar ratio of 2:1. Based on steady-state fluorescence quenching measurements an association constant of 2.4 +/- 0.2 x 10(5) M-1 at 25 degrees C was obtained for the interaction of phosvitin with SHMT. The temperature dependency of the association constant in the range 15-35 degrees C suggests the involvement of ionic forces in the interaction. The thermal inactivation of SHMT followed first order kinetics. In the presence of phosvitin the rate constant decreased and half time increased. The circular dichroism measurements suggest that phosvitin interaction does not involve pyridoxal phosphate binding domain of the enzyme. Although minor changes in the secondary structure of the enzyme were observed, the environment around aromatic amino acids did not change significantly.

Inhibition of Fe(II) catalyzed linoleic acid oxidation and DNA damage by phosvitin.

The oxidation of linoleic acid catalyzed by Fe(II) is strongly inhibited by phosvitin, while chelation with EDTA, NTA or deferoxamine produced only partial inhibition. Interestingly, the DNA degradation catalyzed by Fe(II) in the presence of H2O2 is also inhibited by phosvitin or deferoxamine. In contrast, chelation of the metal ion with EDTA or NTA enhanced the DNA degradation. The results suggest that the nature of interaction between the metal ion and the complexing agent may be an important factor in the generation of active oxygen intermediates.

Evidence for beta-galactosidase catalyzed hydrolysis of paranitrophenyl-beta-D-galactopyranoside anchored in cyclodextrins.

The absorption maximum of p-nitrophenol upon mixing with molar equivalents of alpha-cyclodextrin (alpha-CD) or hydroxypropyl-beta-cyclodextrin (HPB) showed nearly 5 nm shift to the longer wavelength region, indicative of complex formation, while beta-cyclodextrin (beta-CD), gamma-cyclodextrin (gamma-CD) and hydroxyethyl-beta-cyclodextrin (HEB) produced only a marginal shift of about 1-2 nm, suggestive of a weaker interaction. It has been shown by circular dichroism spectral studies that the aglycon part of p-nitrophenyl-beta-D-glycoside (PNPG) is also encapsulated by alpha-cyclodextrin. The encapsulated form of PNPG could be hydrolyzed by beta-galactosidase, the temperature and pH-optima for hydrolysis of anchored substrates being essentially identical to that of free substrate. However, small but consistent increase in Km values were obtained for alpha-cyclodextrin-, HEB- and HPB-anchored substrates. The kcat values also registered an increase for the HEB- and HPB-anchored substrates. However, there was no increase in kinetic efficiency (kcat/Km) of enzyme. The inhibition noted at higher concentrations of HEB- and gamma-cyclodextrin-anchored PNPG but not with o-nitrophenyl-beta-D-galactoside (ONPG)-cyclodextrin mixture suggests that PNPG-cyclodextrin complexes were responsible for the inhibition. Taken together, these results suggest that the enzyme catalyses the hydrolysis of anchored substrates.

Soybean lipoxygenase catalysed oxygenation of unsaturated fatty acid encapsulated in cyclodextrin.

The linoleic or arachidonic acid entrapped in cyclodextrin (alpha, beta or gamma) serves as an excellent substrate for soybean lipoxygenase-1 catalysis. At pH 9.0 the Km values for the beta-cyclodextrin encapsulated arachidonic acid, referred herein as encapsulated substrate, and the Tween-20 dispersed substrate were 7.7 microM and 7.5 microM, respectively. However, the Vmax values for alpha- and beta-cyclodextrin solubilized substrates were lower in comparison with the Tween-20 dispersed substrate. Interestingly, the pH-activity profile for the enzyme towards cyclodextrin encapsulated arachidonic acid showed optimum around 7.5, while that towards Tween-20 dispersion showed the expected broad optimum in the alkaline range (8.5-10.0). The activity with encapsulated substrate at pH 7.5 was at least 5-fold higher than that obtained with Tween-20 dispersed substrate at the corresponding pH. Similar results were obtained using linoleic acid. The second order rate constant, Kcat/Km, for the encapsulated substrate was an order of magnitude higher when compared to the Tween-20 dispersed substrate. The plot of v obtained at pH 9.0, against S gave hyperbolic curves for both the encapsulated as well as the Tween-20 dispersed substrates, whereas at pH 7.5, the curve for cyclodextrin encapsulated arachidonic acid appeared initially concave and then at higher concentrations of the substrate sigmoidal. The positional specificity of soybean lipoxygenase remained unaltered, however.

Influence of detergents and pH on the isolation, purification and molecular properties of alpha-galactosidase-C2 from germinating guar (Cyamopsis tetragonolobus).

alpha-Galactosidase was isolated from germinating guar. The extract also contained small amounts of alpha-mannosidase and beta-mannosidase activities. The fractionation of the enzyme extract with ammonium sulphate (75% saturation) resulted in the appearance of all the three enzymes in a floating lipid complex. The inclusion of detergents such as Triton X-100 and sodium deoxycholate in the extraction medium failed to prevent the appearance of these enzymes in the floating lipid complex. However, by using acetone powder of the seedlings, alpha-galactosidase could be sedimented with ammonium sulphate. The presence of detergents in the extraction medium affected the molecular properties of the enzyme. Using a set of carefully selected conditions alpha-galactosidase was purified to apparent homogeneity. Analytical ultracentrifugation and gel filtration studies of the purified enzyme showed association-dissociation phenomenon as a function of pH and temperature. The effect of pH on the association-dissociation indicates the predominance of electrostatic interactions in the association of subunits.

Isolation and characterization of a novel haemoprotein b-559 from Bengal gram (Cicer arietinum).

A haemoprotein was purified to apparent homogeneity from Bengal-gram seeds. The purified protein exhibited an absorption maximum at 412 nm (Soret band) that upon reduction with dithionite gave rise to a shift in the Soret band to 426 nm with concomitant appearance of an alpha-band at 559 nm and a beta-band at 530 nm. In the reduced state the Bengal-gram haemoprotein showed reactivity towards CO, nitrite and hydroxylamine. SDS/polyacrylamide-slab-gel electrophoresis showed that the haemoprotein has Mr 78,000. Gel-filtration and ultracentrifugal analyses suggest that the Bengal-gram haemoprotein is oligomeric in nature. Since it differs from photosynthetic membrane cytochrome b-559 in solubility in buffer, in reactivity towards CO and in molecular size, it appears to be a novel haemoprotein b-559.

Inactivation of the NADH-dependent activities of nitrate reductase by ferrate.

Nitrate reductase (EC 1.6.6.1) from Chlorella vulgaris, a flavin-cytochrome-molybdenum enzyme, catalyses two types of partial reactions: reduction of exogenous cytochrome c by NADH and reduction of nitrate to nitrite by reduced methyl viologen (reduced 1,1'-dimethyl-4,4'-dipyridine dichloride). Ferrate, an analogue of orthophosphate acting on the phosphate-binding region of the enzymes, abolishes the NADH-nitrate reductase as well as the NADH-cytochrome c activities. In addition, the ability of NADH to reduce the endogenous cytochrome b component of the enzyme is also impaired. The reduction of nitrate by reduced methyl viologen is only partially affected. The results indicate that the ferrate primarily disrupts the NADH site.

Effect of albumin on binding and recovery of enzymes in affinity chromatography on Cibacron Blue.

Bovine serum albumin appears to improve the specificity of Cibacron Blue F3GA in affinity chromatography of enzymes which interact with nucleotides. The action of bovine serum albumin may rest in its ability to selectively mask affinity sites in the dye, which are not specific for the nucleotide-binding region of the enzyme, while not seriously impairing binding nor its elution by nucleotides. Thus, the elution of Chlorella nitrate reductase from a Blue Sepharose chromatographic column by its coenzyme, NADH, fails, unless the column is first treated with bovine serum albumin. Such treatment also improves the recovery of some other nucleotide-binding enzymes tested.

Effect of reduced pyridine nucleotides and tungstate on the in vitro insertion of molybdenum into demolybdo-nitrate reductase of Chlorella vulgaris.

Demolybdo-nitrate reductase (cytochrome c reductase) (NADH: acceptor oxidoreductase, EC 1.6.99.3) of Chlorella vulgaris can be activated in vitro to nitrate reductase by insertion of Mo from molybdate into the apoprotein. Evidence is here presented that reduction of the enzyme by reduced pyridine nucleotides inhibits the process of molybdenum insertion. This report also describes the effect of molybdate and tungstate concentration on the activation process. The activation is sigmoidally related to molybdate concentration with a calculated Hill coefficient of NH = 3. At suboptimal molybdate concentrations, tungstate stimulates enzyme activation by molybdate; but at saturating molybdate concentrations, tungstate is inhibitory. These facts are regarded as an indication that molybdate and tungstate are both positive effectors of molybdenum incorporation, but that they are competitors for the active Mo center.

Molybdenum insertion in vitro in demolybdo nitrate reductase of Chlorella vulgaris.

Demolybdo nitrate reductase (also called cyt c reductase) of Chlorella vulgaris has been converted to active nitrate reductase by insertion of Mo from Na2MoO4 in vitro. A procedure is described which consistently gives about 0.3 unit of nitrate reductase from about 6 units of cyt c reductase, a yield of 30% of the maximum expected, if we calculate on a basis of a ratio of 6 to 1 for the cyt c reductase/nitrate reductase of purified normal enzyme. The demolybdoenzyme is incubated for 30 s at 31 degrees C with molybdate and reduced glutathione (GSH) at pH 4.8, and the pH is then raised to 7, and the incubation continued for 20 min. At the acid pH, there must be a partial denaturation or unfolding which permits Mo insertion, with a refolding to active enzyme at the higher pH. The GSH is not essential for activation, but in its absence the yield of active enzyme was about 50% lower. Experiments with labeled GSH showed that no GSH was incorporated into the protein during the activation procedure. Although the enzyme activity measurements suggested that only 30% of the enzyme was activated, measurements with 99Mo showed that there was one Mo incorporated per subunit weight of 90,000. The Km for nitrate of the activated nitrate reductase was identical with the Km for nitrate of the normal enzyme. On gradient centrifugation, activated nitrate reductase, cyt c reductase, and normal nitrate reductase all behaved identically.

Effects of vanadate on the molybdoproteins xanthine oxidase and nitrate reductase: kinetic evidence for multiple site interaction.

The inhibition of the activity of xanthine oxidase by vanadate was strikingly similar to vanadate inhibition of another molybdoprotein nitrate reductase. Although the main catalytic activity of both enzymes was inhibited, the partial NADH oxidase activity associated with these enzymes was stimulated several fold. It appears that the metal ion binds at multiple site in both enzymes. In the absence of any enzymes a combination of vanadium (V) and molybdenum (V) in air was found to oxide NADH rapidly.

Physical studies on assimilatory nitrate reductase from Chlorella vulgaris.

Assimilatory nitrate reductase (EC 1.6.6.1 NADH:nitrate oxidoreductase) from Chlorella vulgaris purified by affinity chromatography was found to be homogeneous as judged by electrophoresis on sodium dodecyl sulfate-polyacrylamide gel and by analytical ultracentrifugal techniques. The molecular weight of the intact enzyme and that of the enzyme dissociated in 6 M GuHCl, determined by sedimentation equilibrium studies, were 280,000 +/- 10,000 and 90,000 +/- 5,000, respectively. Comparable values were obtained using the S20,w value and the D20,w values in Svedberg's equation. The D20,w values were determined by laser light-scattering measurements. Active enzyme centrifugation showed that the monomer is an active species. A quantitative re-evaluation of the prosthetic groups present (FAD, heme, and molybdenum) was also made and was consistent with the conclusion that the active monomer contains three subunits as previously deduced by Solomonson et al. ((1975) J. Biol. Chem. 250, 4120). Electron micrographs showed images which corresponded to three subunits, supporting the data obtained by hydrodynamic studies. The enzyme is not cigar-shaped, as previously surmised, but has a roughly globular structure.

The effect of vanadium on nitrate reductase of Chlorella vulgaris.

Added vanadate ions inhibit purified nitrate reductase from Chlorella vulgaris by reacting with the enzyme in a manner rather similar to that of HCN. Thus vanadate, like HCN, forms an inactive complex with the reduced enzyme, and this inactivated enzyme can be reactivated rapidly by adding ferricyanide. The inactive vanadate enzyme complex is less stable than the inactive HCN complex, and the two can be distinguished by the fact that EDTA causes a partial reactivation of the former, but not of the latter. Vanadate can also cause an increase in HCN formation by intact Chlorella vulgaris cells. When these cells were incubated with vanadate, their nitrate reductase was reversibly inactivated, and all of this inactive enzyme could be shown to be the HCN complex rather than the vanadate complex. When HCN and vanadate are both present, the HCN-inactivated enzyme, being more stable, will be formed in preference to the vanadate-inactivated enzyme.

Regulatory enzymes of carbohydrate and energy metabolism in the rabbit blastocyst.

The activities of phosphofructokinase, pyruvate kinase, citrate synthase and creatine kinase were determined in blastocysts from rabbits at 144 h post coitum and in similar blastocysts cultured for 24 h with or without oestradiol-17beta (1 microgrm/ml). There was a significant increase in all the enzymes during the 24-h culture period but oestradiol had no effect.

Biosynthesis of a prostaglandin by a plant enzyme.

Lipoxygenase-2 from soybeans catalyzes the oxygenation of arachidonic acid to form significant amounts of a prostaglandin product. Results obtained with the dithionite-reduced derivatized product upon combined gas chromatography-mass spectroscopy are consistent with structures of the stereoisomers of 9,11,15-trihydroxyprosta-5,13-dienoic acid. A portion of the material formed by reduction of the enzyme product with dithionite reacts positively in a radioimmunoassay against rabbit anti-prostaglandin F2alpha antibody. This appears to be the first report of the synthesis of a prostaglandin by a non-animal enzyme.