Sheldon W. May

Applications of oxidoreductases.

Oxidoreductases comprise the large class of enzymes that catalyze biological oxidation/reduction reactions. Because many chemical and biochemical transformations involve oxidation/reduction processes, developing practical biocatalytic applications of oxidoreductases has long been an important goal in biotechnology. During the past year, significant progress has been made in the development of oxidoreductase-based diagnostic tests and improved biosensors, in the design of innovative systems for regeneration of essential coenzymes, in the construction bioreactors for biodegradation of pollutants and for biomass processing, and in the development of oxidoreductase-based approaches for synthesis of polymers and oxyfunctionalized organic substrates.

Selenium-based pharmacological agents: an update.

The biochemistry and pharmacology of selenium is a subject of intense current interest, particularly from the viewpoint of public health. Selenium, long recognised as a dietary antioxidant, is now known to be an essential component of the active sites of several enzymes, including glutathione peroxidase and thioredoxin reductase, which catalyse reactions essential to the protection of cellular components against oxidative and free radical damage. A low concentration of selenium in plasma has been identified as a risk factor for several diseases, including cancer, cardiovascular disease, osteoarthritis and AIDS, and several large-scale selenium supplementation human trials are now underway. Evidence is emerging that, at least in the case of cancer, the antitumorigenic effect of selenium supplementation arises at least in part from enhanced production of specific selenium-containing metabolites, not just from maximal expression of selenoenzymes. Therefore a number of novel pharmaceutical agents which are selenium-based or which target specific aspects of selenium metabolism are under development. Among these are orally-active antihypertensive agents, anticancer, antiviral, immunosuppressive and antimicrobial agents, and organoselenium compounds which reduce oxidative tissue damage and edema. It can be anticipated that as our understanding of the basic biology and biochemistry of selenium increases, the coming years will bring further development of new selenium-based pharmaceutical agents with therapeutic potential toward a variety of human diseases.

Low-flow interface for liquid chromatography–inductively coupled plasma mass spectrometry speciation using an oscillating capillary nebulizer

The application of a novel nebulizer, the oscillating capillary nebulizer (OCN), is described for use in speciation studies. The nebulizer has certain features which make it very suitable for this application, without modification, at both micro-flows (1 µl min–1) and macro-flows (1 ml min–1). Short- and long-term precision at typical operating flows are comparable to a normal (1 ml min–1) concentric glass nebulizer. Column-to-nebulizer dead volume is approximately 1 µl. The narrow drop size distribution for the nebulizer at low flows leads to excellent sensitivity when coupled to a micro-LC column. Post-column peak broadening introduced by the interface is minor at flows 5 µl min–1, but widens the peaks noticeably at flows between 1 and 5 µl min–1. The very high efficiency of the nebulizer at flows <50 µl min–1 is exemplified by the fact that no drain is necessary at these flows in the open spray chamber, as no visible liquid condenses on the chamber walls. The ICP-MS response for the OCN (counts per ng of Se injected) does not change when water is replaced by methanol as solvent, whereas with a conventional nebulizer, a solvent change of this type inevitably results in a significant change in response. The OCN was used for the reversed-phase LC separation of a mixture of five organic Se compounds of pharmacological significance, at flows of 12, 50 and 400 µl min–1. With use of a 0.5 mm id column, a flow rate of 12 µl min–1 and a 60 nl injection, good peak separation was found, with an average efficiency of ≈ 10000 plates and a detection limit of around 30 pg.

Structural effects on the reactivity of substrates and inhibitors in the epoxidation system of Pseudomonas oleovorans

The epoxidation reaction catalyzed by an enzyme system of Pseudomonas oleovorans exhibits a substrate specificity different from that expected on the basis of chemical reactivity in non-enzymatic epoxidation reactions. Cyclic and internal olefins, aromatic compounds and styrene are not epoxidated. The reactivity of straight chain diolefins is maximal for octadiene and falls off rapidly as the carbon chain is shortened, but decreases only slightly as the chain is lengthened. In contrast, methyl group hydroxylation is less sensitive to decreasing chain length. As a consequence, propylene and 1-butene are hydroxylated but not epoxidated by this enzyme system. With the substrate 1-decene, which is capable of undergoing both epoxidation and hydroxylation, the former reaction predominates. Methyl imidoesters were found to be inhibitors of enzymatic epoxidation, and the potency of a homologous series of imidoester inhibitors was examined. The results parallel the substrate specificity patterns observed, and support the conclusion that the mode of substrate binding severely moderates the inherent chemical reactivity of the activated oxygen in this system. The effect of the bifunctional imidoester, dimethyladipimidate, was also examined and the results compared with those obtained in other investigations.

Enzymatic epoxidation reactions

Abstract The oxygenases - enzymes which incorporate molecular oxygen directly into organic molecules - are ubiquitous and of high metabolic significance. These enzymes play crucial roles in the degradation of drugs and foreign substances and in the biosynthesis, interconversion and degradation of amino acids, lipids, porphyrins, vitamins and hormones. Thus, they are centrally involved in the mechanisms of cytotoxicity, mutagenicity, carcinogenicity and tissue necrosis. From the standpoint of enzyme technology, the ability of these enzymes to incorporate molecular oxygen into organic substrates efficiently and selectively is highly enticing, since such reactions are poorly accomplished using conventional chemistry. This review focuses on enzymatic epoxidation reactions, one example of the many chemical transformations catalysed by oxygenases. By way of introduction, an overview of the role of enzymatic epoxidation reactions in the metabolism of polycyclic aromatic hydrocarbons, in steroid biosynthesis and interconversion, and in various other pathways is presented. Following this, enzymatic epoxidation of simple olefins is considered in detail, with emphasis on bacterial systems and discussion of both enzymology and reactivity characteristics. Finally, a number of major issues which must be confronted if complex oxygenase systems are to be utilized in enzyme technology application are briefly discussed. Among these are specialized immobilization techniques, cofactor recycling, problems of enzyme stability, and the intriguing possibility of utilizing mechanistic information in the design of non-enzymatic, chemical model systems which mimic oxygenase catalysis .

A resonance Raman study of substrate and inhibitor binding to protocatechuate-3,4-dioxygenase.

Abstract Resonance Raman spectra were obtained for complexes of protocatechuate-3,4-dioxygenase with substrate and hydroxybenzoate inhibitors. The data establish metal coordination by these bound species and demonstrate further that tyrosine ligation, present in the resting enzyme, is not altered in the complexes. For the inhibitors, 3-chloro-4-hydroxybenzoate and 3-fluoro-4-hydroxybenzoate, the data are interpreted as indicating iron ligation by the phenolate functionality. For the substrate, 3,4-dihydroxyphenylproprionate, chelation via the o -dihydroxy grouping is proposed. In all three complexes tyrosine ligands present in the resting enzyme are not displaced. The inhibitor scattering intensity was utilized as an internal standard to estimate that two tyrosines are coordinated to the iron at the active site.

Pyruvate-extended Amino Acid Derivatives as Highly Potent Inhibitors of Carboxyl-terminal Peptide Amidation

Carboxyl-terminal amidation, a required post-translational modification for the bioactivation of many neuropeptides, entails sequential enzymatic action by peptidylglycine monooxygenase (PAM, EC 1.14.17.3) and peptidylamidoglycolate lyase (PGL, EC 4.3.2.5). The monooxygenase, PAM, first catalyzes conversion of a glycine-extended pro-peptide to the corresponding α-hydroxyglycine derivative, and the lyase, PGL, then catalyzes breakdown of this α-hydroxyglycine derivative to the amidated peptide plus glyoxylate. We now introduce the first potent inhibitors for peptidylamidoglycolate lyase. These inhibitors, which can be viewed as pyruvate-extended N-acetyl amino acids, constitute a novel class of compounds. They were designed to resemble likely transient species along the reaction pathway of PGL catalysis. A general synthetic procedure for preparation of pyruvate-extended N-acetyl amino acids or peptides is described. Since these compounds possess the 2,4-dioxo-carboxylate moiety, their solution tautomerization was investigated using both NMR and high performance liquid chromatography analyses. The results establish that freshly prepared solutions of N-Ac-Phe-pyruvate consist predominantly of the enol tautomer, which then slowly tautomerizes to the diketo form when left standing for several days in an aqueous medium; upon acidification, formation of the hydrate tautomer occurs. Kinetic experiments established that these novel compounds are highly potent, pure competitive inhibitors of PGL. Kinetic experiments with the ascorbate-dependent copper monooxygenases, PAM and dopamine-β-monooxygenase, established that these compounds also bind competitively with respect to ascorbate; however, pyruvate-extended N-acyl-amino acid derivatives possessing hydrophobic side chains are much more potent inhibitors of PGL than of PAM. Selective targeting of N-Ac-Phe-pyruvate so as to inhibit the lyase, but not the monooxygenase, domain was demonstrated with the bifunctional amidating enzyme of Xenopus laevis. The availability of potent inhibitors of PGL should facilitate studies regarding the possible biological role of α-hydroxyglycine-extended peptides.

Dopamine-B-hydroxylase: Suicide inhibition by the novel olefinic substrate, 1-phenyl-1-aminomethylethene

Dopamine-B-hydroxylase [E.C.1.14.17.1] plays a key role in the biosynthetic interconversion of neurotransmitters. It is now demonstrated for the first time that dopamine-B-hydroxylase also catalyzes the oxygenation of an olefinic substrate, 1-phenyl-1-aminomethylethene, producing 2,3-dihydroxy-2-phenylpropylamine after acid workup. This reaction gives the normal oxygenase stoichiometry of electrons to O2 to product of 2:1:1, and is kinetically comparable to other oxygenase activities of dopamine-B-hydroxylase, with a kcat value of 10 sec-1 and a KM of 8.3 mM. 1-Phenyl-1-aminomethylethene is also a time-dependent, first-order inactivator of dopamine-B-hydroxylase. The inactivation process exhibits the characteristics of mechanism-based, irreversible inactivation, giving a KI value of 13 mM and a kinac of 0.04 min-1. The central role of dopamine-B-hydroxylase in catecholamine metabolism suggests possible pharmacological uses for olefinic inhibitors of this enzyme.

Iso-coenzyme A

Iso-coenzyme A is an isomer of coenzyme A in which the monophosphate is attached to the 2′-carbon of the ribose ring. Although iso-CoA was first reported in 1959 (Moffatt, J. G., and Khorana, H. G. (1959) J. Am. Chem. Soc. 81, 1265–1265) to be a by-product of the chemical synthesis of CoA, relatively little attention has been focused on iso-CoA or on acyl-iso-CoA compounds in the literature. We now report structural characterizations of iso-CoA, acetyl-iso-CoA, acetoacetyl-iso-CoA, and β-hydroxybutyryl-iso-CoA using mass spectrometry (MS), tandem MS, and homonuclear and heteronuclear NMR analyses. Although the 2′-phosphate isomer of malonyl-CoA was recently identified in commercial samples, previous characterizations of iso-CoA itself have been based on chromatographic analyses, which ultimately rest on comparisons with the degradation products of CoA and NADPH or have been based on assumptions regarding enzyme specificity. We describe a high performance liquid chromatography methodology for separating the isomers of several CoA-containing compounds. We also report here the first examples of iso-CoA-containing compounds acting as substrates in enzymatic acyl transfer reactions. Finally, we describe a simple synthesis of iso-CoA from CoA, which utilizes β-cyclodextrin to produce iso-CoA with high regioselectivity, and we demonstrate a plausible mechanism that accounts for the existence of iso-CoA isomers in commercial preparations of CoA-containing compounds. We anticipate that these results will provide methodology and impetus for investigating iso-CoA compounds as potential pseudo-substrates or inhibitors of the >350 known CoA-utilizing enzymes.

Peptide amidating enzymes are present in cultured endothelial cells

Carboxy-terminal amidation is a prevalent post-translational modification necessary for the bioactivity of many peptides. We now report that the two enzymes essential for amidation, peptidylglycine alpha-monooxygenase (PAM) and peptidylamidoglycolate lyase (PGL), are present in both the cytosol and membrane fractions of cultured bovine aortic endothelial cells. Endothelial PAM exhibits ascorbate-dependent turnover and is inactivated by the mechanism-based inactivator, 4-phenyl-3-butenoic acid (PBA), whereas PGL activity is independent of ascorbate and is not affected by PBA. These enzymological characteristics correspond to those of amidating enzymes from other tissues. These results suggest a heretofore unrecognized role for alpha-amidated peptides in cardiovascular function.