Stereoselective hydrogen abstraction in leukotriene A4 synthesis by purified 5-lipoxygenase of porcine leukocytes

Arachidonate 5-lipoxygenase purified from porcine leukocytes transformed arachidonic acid to 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid. By the leukotriene A synthase activity of the same enzyme the product was further metabolized to leukotriene A4 (actually detected as 6-trans-leukotriene B4, 12-epi-6-trans-leukotriene B4, and 5,6-dihydroxy-7,9,11,14-eicosatetraenoic acids). The enzyme was incubated with [10-DR-3H]- or [10-LS-3H]-labeled arachidonic acid, and 6-trans-LTB4 and its 12-epimer were analyzed. More than 90% of 10-DR-hydrogen was lost while about 100% of 10-LS-hydrogen was retained, indicating a stereospecific hydrogen elimination from C-10 during the formation of leukotriene A4.     

Stereoselective hydrogen abstraction in leukotriene A4 synthesis by purified 5-lipoxygenase of porcine leukocytes

Arachidonate 5-lipoxygenase purified from porcine leukocytes transformed arachidonic acid to 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid. By the leukotriene A synthase activity of the same enzyme the product was further metabolized to leukotriene A₄ (actually detected as 6-trans-leukotriene B₄, 12-epi-6-trans-leukotriene B₄, abd 5,6-duhydroxy-7,9,11,14-eicosatetraenoic acids). The enzyme was incubated with [10-D_R-³H]- or [10-L_S-³H]- labeled arachidonic acid, and 6-trans-LTB₄ and its 12-epimer were analyzed. More than 90% of 10-D_R-hydrogen was lost while about 100% of 10-L_S-hydrogen was retained, indicating a stereospecific hydrogen elimination from C-10 during the formation of leukotriene A₄.     

Pyridoxamine-5'-phosphate oxidase exhibits no specificity in prochiral hydrogen abstraction from substrate

The stereochemistry for hydrogen removal from pyridoxamine 5'-phosphate with liver pyridoxine (pyridoxamine)-5'-phosphate oxidase was examined to determine whether or not there are significant steric constraints at the substrate region of the active site of the oxidase. For this, pyridoxal 5'-phosphate was reduced with tritium-labeled sodium borohydride in ammoniacal solution to yield racemically labeled [4',4'-3H]pyridoxamine 5'-phosphate which was then chemically or enzymatically oxidized to [4'-3H]pyridoxal 5'-phosphate. This latter was used as coenzyme with either L-aspartate (L-glutamate) aminotransferase and L-glutamate or L-glutamate decarboxylase and alpha-methyl-DL-glutamate to generate [4'-3H]pyridoxamine 5'-phosphate known to be labeled in the R-position. Reaction of the oxidase with the pro-R as well as the pro-R,S-labeled substrates followed by isolation of [4'-3H]pyridoxal 5'-phosphate and 3H2O revealed only half the radioactivity was abstracted from the original substrate in either case. Hence, the oxidase is not stereospecific and equally well catalyzes removal of either pro-R or pro-S hydrogen from the 4-methylene of pyridoxamine 5'-phosphate.     

Expression of recombinant galactose oxidase by Pichia pastoris

Galactose oxidase catalyzes the oxidation of a variety of primary alcohols, producing hydrogen peroxide as a product. Among hexose sugars, the enzyme exhibits a high degree of specificity for the C6-hydroxyl of galactose and its derivatives, underlying a number of important bioanalytical applications. Galactose oxidase cDNA has been cloned for expression in Pichia pastoris both as the full-length native sequence and as a fusion with the glucoamylase signal peptide. Expression of the full-length native sequence results in a mixture of partly processed and mature galactose oxidase. In contrast, the fusion construct directs efficient secretion of correctly processed galactose oxidase in high-density, methanol-induced fermentation. Culture conditions (including induction temperature and pH) have been optimized to improve the quality and yield (500 mg/L) of recombinant enzyme. Lowering the temperature from 30 to 25 degrees C during the methanol induction phase results in a fourfold increase in yield. A simple two-step purification and one-step activation produce highly active galactose oxidase suitable for a wide range of biomedical and bioanalytical applications.     

Oxidation of glycolipids in liposomes by galactose oxidase

Small unilamellar phosphatidylcholine vesicles containing globo-series glycolipids were labeled by the galactose oxidase/NaB[3H]4 procedure. The major glycolipid of human red cells, globoside, was the best substrate for galactose oxidase both in vesicles and in tetrahydrofuran-containing buffer. The oxidation rates of membrane-bound ceramide trihexoside and Forssman glycolipid were one-fourth and one-tenth, respectively, of the oxidation rate of globoside. Membrane-bound ceramide dihexoside was not a substrate for galactose oxidase, although it was readily oxidized in tetrahydrofuran-containing buffer. Soluble sialoglycoproteins and membrane-incorporated glycophorin A stimulated the oxidation of globoside-containing vesicles, whereas membrane-bound GD1a ganglioside had no effect on globoside oxidation.     

Cofactor processing in galactose oxidase

GO (galactose oxidase; E.C. 1.1.3.9) is a monomeric 68 kDa enzyme that contains a single copper ion and an amino acid-derived cofactor. The enzyme is produced by the filamentous fungus Fusarium graminearum as an extracellular enzyme. The enzyme has been extensively studied by structural, spectroscopic, kinetic and mutational approaches that have provided insight into the catalytic mechanism of this radical enzyme. One of the most intriguing features of the enzyme is the post-translational generation of an organic cofactor from active-site amino acid residues. Biogenesis of this cofactor involves the autocatalytic formation of a thioether bond between Cys-228 and Tyr-272, the latter being one of the copper ligands. Formation of this active-site feature is closely linked to the loss of an N-terminal 17 amino acid prosequence. When copper and oxygen are added to this pro-form of GO (pro GO), purified in copper-free conditions from the heterologous host Aspergillus nidulans, mature GO is formed by an autocatalytic process. Structural comparison of pro GO with mature GO reveals overall structural similarity, but with some regions showing significant local differences in main-chain position. Some side chains of the active-site residues differ significantly from their positions in the mature enzyme. These structural effects of the prosequence suggest that it may act as an intramolecular chaperone to provide an open active-site structure conducive to copper binding and chemistry associated with cofactor formation. The prosequence is not mandatory for processing, as a recombinant form of GO lacking this region and purified under copper-free conditions can also be processed in an autocatalytic copper- and oxygen-dependent manner.     

Catalytic reaction profile for alcohol oxidation by galactose oxidase

Galactose oxidase is a remarkable enzyme containing a metalloradical redox cofactor capable of oxidizing a variety of primary alcohols during enzyme turnover. Recent studies using 1-O-methyl alpha-D-galactopyranoside have revealed an unusually large kinetic isotope effect (KIE) for oxidation of the alpha-deuterated alcohol (kH/kD = 22), demonstrating that cleavage of the 6,6'-di[2H]hydroxymethylene C-H bond is fully rate-limiting for oxidation of the canonical substrate. This step is believed to involve hydrogen atom transfer to the tyrosyl phenoxyl in a radical redox mechanism for catalysis [Whittaker, M. M., Ballou, D. P., and Whittaker, J. W. (1998) Biochemistry 37, 8426-8436]. In the work presented here, the enzyme's unusually broad substrate specificity has allowed us to extend these investigations to a homologous series of benzyl alcohol derivatives, in which remote (meta or para) substituents are used to systematically perturb the properties of the hydroxyl group undergoing oxidation. Quantitative structure-activity relationship (QSAR) correlations over the steady state rate data reveal a shift in the character of the transition state for substrate oxidation over this series, reflected in a change in the magnitude of the observed KIE for these reactions. The observed KIE values have been shown to obey a log-linear correlation over the substituent parameter, Hammett sigma. For the relatively difficult to oxidize nitro derivative, the KIE is large (kH/kD = 12.3), implying rate-limiting C-H bond cleavage for the oxidation reaction. This contribution becomes less important for more easily oxidized substrates (e.g., methoxy derivatives) where a much smaller KIE is observed (kH/kD = 3.6). Conversely, the solvent deuterium KIE is vanishingly small for 4-nitrobenzyl alcohol, but becomes significant for the 4-methoxy derivative (kH2O/kD2O = 1.2). These experiments have allowed us to develop a reaction profile for substrate oxidation by galactose oxidase, consisting of three components (hydroxylic proton transfer, electron transfer, and hydrogen atom transfer) comprising a single-step proton-coupled electron transfer mechanism. Each component exhibits a distinct substituent and isotope sensitivity, allowing them to be identified kinetically. The proton transfer component is unique in being sensitive to the isotopic character of the solvent (H2O or D2O), while hydrogen atom transfer (C-H bond cleavage) is independent of solvent composition but is sensitive to substrate labeling. In contrast, electron transfer processes will in general be less sensitive to isotopic substitution. Our results support a mechanism in which initial proton abstraction from a coordinated substrate activates the alcohol toward inner sphere electron transfer to the Cu(II) metal center in an unfavorable redox equilibrium, forming an alkoxy radical which undergoes hydrogen atom abstraction by the tyrosine-cysteine phenoxyl free radical ligand to form the product aldehyde.     

Stereospecific oxidation of aliphatic alcohols catalyzed by galactose oxidase

The stereospecificity of galactose oxidase (EC 1.1.3.9) from Dactylium dendroides in the oxidation of simple three-carbon alcohols has been examined. The enzyme oxidizes glycerol to optically pure S(?)glyceraldehyde. In addition to this prochiral stereospecificity, galactose oxidase also exhibits enantiomeric stereospecificity in the oxidation of 3-halo-1,2-propanediols: the R isomer appears to be a better substrate than its S counterpart. The above stereochemistry of galactose oxidase-catalyzed oxidation of “unnatural” substrates, non-sugar alcohols, can be predicted on the basis of the conformation of the natural substrate of the enzyme, D-galactose.     

Transformation of human peripheral lymphocytes by galactose oxidase.

Human peripheral lymphocytes can be transformed by treatment with galactose oxidase alone. Prior treatment with neuraminidase enhances this effect. The aldehyde blocking agents thiocarbohydrazide, hydroxylamine, dimedone, and sodium borohydride block transformation when they follow, but not when they precede, galactose oxidase treatment. Thus, as is the case for periodate-induced lymphocyte transformation, the formation of free aldehyde at the cell surface would seem to be a critical event in the triggering of transformation by this agent. The degree of transformation is highly variable from individual to individual, and also for the same donor at different times. However, the lymphocytes of some people give a consistently poor response to galactose oxidase. Similar results have been obtained for periodate-induced transformation of human lymphocytes, but to this date this is unexplained.     

Oxidation rates of some desialylated glycoproteins by galactose oxidase

Patterns of oxidation of dilute solutions of desialylated fetuin and submaxillary mucin by galactose oxidase have been examined. A significant portion (20-40%) of the terminal galactosyls exposed on the glycoproteins, which theoretically were expected to be accessible to the enzyme, was not oxidized. In comparison, galactosyls in oligosaccharides released from completely desialylated glycoproteins were oxidized more effectively with an apparently lower degree of crypticity to the enzyme. Partial desialylation usually resulted in a reduction of both the rate and the final level of substrate oxidation. A second cycle of oxidation of a desialylated substrate earlier oxidized by galactose oxidase and then reduced by NaB3H4 revealed a selectivity in the pattern of galactosyl oxidation. The same galactosyl residues oxidized in the first cycle were again the most susceptible to oxidation in the second cycle, leaving unmodified the same fraction of galactosyls throughout both cycles. The relevance of these results to the application of the galactose oxidase-NaBH4 procedure for detecting and measuring desialylated glycoconjugates in solution and in biological membranes is discussed.     

Properties of Fusarium graminearum galactose oxidase

The kinetics and action mechanism of the galactose oxidase from Fusarium graminearum were studied. pH-optimum of the enzyme activity and stability was 7.0, the activity and stability of the galactose oxidase being decreased at any other values of pH. The enzyme is destabilized at acidic pH that is connected with protonization of its ionogenic group with pK 4.7. The temperature optimum of the galactose oxidase is 35 degrees C. When studying the enzyme thermoinactivation, it was found that at temperatures below 30 degrees C the energy of activation of denaturation was about 40 kcal/mole and at temperatures ranging from 30 to 70 degrees C - 13 kcal/mole. On the basis of the data obtained it was concluded that a low-temperature form of the galactose oxidase, possessing a higher energy of activation of denaturation, is more active than a high-temperature form. The value of Km for the enzyme in respect to galactose was 0.19 M, and the value of Vmax = 360 mumole/min per g of the preparation.     

The radical chemistry of galactose oxidase

Galactose oxidase is a free radical metalloenzyme containing a novel metalloradical complex, comprised of a protein radical coordinated to a copper ion in the active site. The unusually stable protein radical is formed from the redox-active side chain of a cross-linked tyrosine residue (Tyr-Cys). Biochemical studies on galactose oxidase have revealed a new class of oxidation mechanisms based on this free radical coupled-copper catalytic motif, defining an emerging family of enzymes, the radical-copper oxidases. Isotope kinetics and substrate reaction profiling have provided insight into the elementary steps of substrate oxidation in these enzymes, complementing structural studies on their active site. Galactose oxidase is remarkable in the extent to which free radicals are involved in all aspects of the enzyme function: serving as a key feature of the active site structure, defining the characteristic reactivity of the complex, and directing the biogenesis of the Tyr-Cys cofactor during protein maturation.     

The stereospecificity of hydrogen abstraction by uridine diphosphoglucose dehydrogenase

UDP-glucose dehydrogenase catalyzes the incorporation of tritium into UDP-glucose (UDPG) in the presence of UDP-α-D-gluco-hexodialdose (UDP-Glc-6-CHO) and [B-³H]-NADH. The ³H is located exclusively at C-6 of the glucose moiety of UDPG and at least 79% of it is in the pro-R position. It is concluded that UDPG dehydrogenase catalyzes the abstraction of the pro-R hydrogen at C-6 of the glucose moiety of the substrate as the first step in the conversion of UDPG to UDP-glucuronic acid. The apparent lack of complete stereospecificity has been shown to result from a hitherto undetected reversible redox reaction prior to the release of UDP-glucuronic acid by the enzyme.     

Quantitation of galactocerebrosides and sulfatides by galactose oxidase and sodium borotritide

A method has been developed for the quantitation of galactocerebrosides using galactose oxidase and sodium borotritide that is highly specific for terminal galactose or galactosamine containing glycolipids. Prior separation of galactocerebrosides from other lipids is unnecessary and nanomole quantities of galactocerebrosides can be reliably estimated within 6–7 h. This method can be adapted with slight modification for quantitation of sulfatides in total lipid extract.     

Continuous indirect electrochemical regeneration of galactose oxidase

The development of an indirect anaerobic electrochemical regeneration of galactose oxidase (GOase) allows the prevention of the undesired production of the enzyme inhibitor hydrogen peroxide, which is generated under aerobic regeneration conditions during synthetic applications of GOase. The pH optimum for the electrochemical regeneration of GOase with polyethyleneglycol-modified ferrocene mediators in carbonate buffer is 10.8. Total turnover numbers achieved by either electrochemical or aerobic regeneration of GOase are almost the same. The electrochemical regeneration is half as fast as the aerobic regeneration. It is not necessary to work under anaerobic conditions, because at pH 10.8 the aerobic regeneration of GOase is prevented. The enzyme can be stabilized most effectively by immobilization on an aminopropylated polysiloxane (DELOXAN) via the glutaric dialdehyde procedure with good activity yields up to 37%. Buffers containing amino groups proved to be fatal for long-term GOase stability.