Structural identification and synthesis of luciferin from the bioluminescent earthworm, Diplocardia longa.

For the first time, luciferin from a bioluminescent earthworm has been purified, identified, and synthesized. This luciferin from the North American species, Diplocardia longa, is a simple aldehyde compound, N-isovaleryl-3-aminopropanal, with an amide functional group. It is a clear, odorless oil at room temperature. It is nonvolatile and has no near-uv-visible absorption or fluorescence. Derivatives of this compound were made to facilitate its identification: the luciferin 2,4-dinitrophenylhydrazone (mp 174 degrees C), a yellow crystalline solid; and the luciferin alcohol, a clear oil. Synthesis of Diplocardia luciferin yielded an oil of identical spectroscopic (proton nuclear magnetic resonance (NMR), 13C NMR, mass, and ir), chemical (dinitrophenylhydrazone and alcohol derivatives, bioluminescence activity), and physical (thin-layer chromatography, volatility) properties to those of the purified native Diplocardia luciferin.     

Detoxification of sulfur mustard by enzyme-catalyzed oxidation using chloroperoxidase

One of the most interesting methods for the detoxification of sulfur mustard is enzyme-catalyzed oxidation. This study examined the oxidative destruction of a sulfur mustard by the enzyme chloroperoxidase (EC 1.11.1.10). Chloroperoxidase (CPO) belongs to a group of enzymes that catalyze the oxidation of various organic compounds by peroxide in the presence of a halide ion. The enzymatic oxidation reaction is affected by several factors: pH, presence or absence of chloride ion, temperature, the concentrations of hydrogen peroxide and enzyme and aqueous solubility of the substrate. The optimum reaction conditions were determined by analyzing the effects of all factors, and the following conditions were selected: solvent, Britton–Robinson buffer (pH = 3) with tert-butanol (70:30 v/v); CPO concentration, 16 U/mL; hydrogen peroxide concentration, 40 mmol/L; sodium chloride concentration, 20 mmol/L. Under these reaction conditions, the rate constant for the reaction is 0.006 s⁻¹. The Michaelis constant, a measure of the affinity of an enzyme for a particular substrate, is 1.87 × 10⁻³ M for this system. The Michaelis constant for enzymes with a high affinity for their substrate is in the range of 10⁻⁵ to 10⁻⁴ M, so this value indicates that CPO does not have a very high affinity for sulfur mustard.     

A postulated mechanism for the bioluminescent oxidation of reduced flavin mononucleotide

A mechanism is presented for the luciferase catalyzed oxidation of reduced flavin mononucleotide with oxygen in the presence of long-chain aldehyde. The mechanism involves the formation of a flavin peroxy anion which attacks aldehyde. A Baeyer-Villiger type shift leads to oxidation of aldehyde to acid, and to formation of hydroxide and excited protonated flavin which emits a photon. The mechanism is consistent with known details of the bioluminescent reaction and with known reactions of flavins and allows several verifiable predictions to be made.     

Renilla luciferin as the substrate for calcium induced photoprotein bioluminescence. Assignment of luciferin tautomers in aequorin and mnemiopsin.

A study was made of the effects of pH and protic and aprotic solvents on the spectral properties of Renilla (sea pansy) luciferin and a number of its analogs. The results have made possible the assignment of two tautomeric forms of Renilla luciferin, one which absorbs maximally at 435 nm and another which exhibits an absorption maximum at 454 nm. Furthermore the results provide an explanation for the visible absorption characteristics of the photoproteins aequorin (lambda-max 454 nm) and mnemiopsin (lambda-max 435 nm). In addition a Renilla-like luciferin can be extracted from both of these photoproteins. This luciferin produces light with Renilla luciferase, at a rate dependent upon the concentration of dissolved oxygen, and in other respects is indistinguishable from Renilla luciferin in this bioluminescent reaction. The results suggest that the native chromophore in both photoproteins is Renilla luciferin (or a nearly identical derivative). The results also suggest that a hydroperoxide intermediate probably exists in photoproteins, on energetic grounds, and to account for the oxygen concentration independency of the rate of photoprotein reactions. This hydroperoxide may be attached initially to an amino-acid side chain (possibly indolyl-OOH, imidazoyl-OOH, or -SOOH) rather than to the luciferin chromophore.     

Mechanism of L-serine oxidation in Entamoeba histolytica.

1. The enzymatic mechanism of oxygen uptake elicited by L-serine in axenically cultivated trophozoites of Entamoeba histolytica was investigated. 2. Of 22 amino acids examined, only L-serine stimulated oxygen consumption by intact and disrupted amoebae. 3. Pyruvate, a product of serine metabolism, also stimulated oxygen consumption in the amoebae. 4. Characterization of the oxygen uptake elicited by both L-serine and pyruvate, and analysis of the products of L-serine metabolism indicate that the amino acid is first converted to pyruvate. 5. L-Serine dehydratase, which catalyzes the deamination of serine to pyruvate, was detected primarily in the soluble fraction of the amoebae. D-Serine potently inhibited the enzyme, as well as oxygen uptake in the presence of L-serine but not in the presence of pyruvate. 6. The pyruvate formed is oxidized, at least in part, by a novel pyruvate oxidase involving the uptake of molecular oxygen.     

Calorimetry of enzyme-catalyzed reactions

This mini-review shows the valuable contributions of Professor Julian Sturtevant to the current applications of calorimetry to the study of enzyme-catalyzed reactions. The more recent applications of calorimetric techniques such as isothermal titration calorimetry and flow calorimetry to the study of enzyme kinetics, as well as the advantages on using calorimetric techniques in the determination of kinetic parameters of enzymes, is also discussed here.