Mechanism of the reaction catalyzed by DL-2-haloacid dehalogenase as determined from kinetic isotope effects.

dl-2-Haloacid dehalogenase from Pseudomonas sp. 113 is a unique enzyme because it acts on the chiral carbons of both enantiomers, although its amino acid sequence is similar only to that of d-2-haloacid dehalogenase from Pseudomonas putida AJ1 that specifically acts on (R)-(+)-2-haloalkanoic acids. Furthermore, the catalyzed dehalogenation proceeds without formation of an ester intermediate; instead, a water molecule directly attacks the alpha-carbon of the 2-haloalkanoic acid. We have studied solvent deuterium and chlorine kinetic isotope effects for both stereoisomeric reactants. We have found that chlorine kinetic isotope effects are different: 1.0105 +/- 0.0001 for (S)-(-)-2-chloropropionate and 1.0082 +/- 0.0005 for the (R)-(+)-isomer. Together with solvent deuterium isotope effects on V(max)/K(M), 0.78 +/- 0.09 for (S)-(-)-2-chloropropionate and 0.90 +/- 0.13 for the (R)-(+)-isomer, these values indicate that in the case of the (R)-(+)-reactant another step preceding the dehalogenation is partly rate-limiting. Under the V(max) conditions, the corresponding solvent deuterium isotope effects are 1.48 +/- 0.10 and 0.87 +/- 0.27, respectively. These results indicate that the overall reaction rates are controlled by different steps in the catalysis of (S)-(-)- and (R)-(+)-reactants.

Application of triazine "superactive esters" in the repetitive synthesis of oligopeptides. Part 2. Synthesis of N-terminal hexapeptide of emerimicin III.

N-terminal fragment of emerimicin III has been synthesized by the repetitive method in solution, involving triazine ,,superactive esters". The synthetic protocol, equivalent to the classic REMA procedure, has been applied in the step by step approach, and in the fragment coupling affording all the peptide bonds. By monitoring the progress of the synthesis by FAB-MS, 1H-NMR and HPLC, the structure and high purity of the final products have been confirmed.

Application of triazine "superactive esters" in the repetitive synthesis of oligopeptides. Part 1. Synthesis of [54-59] fragment of human beta-casein.

A new synthetic protocol which considerably improves the classic REMA procedure is proposed herein. The offered modification is based on the application of triazine "superactive esters" as the superior substitutes for mixed anhydrides, which have been used as acylating reagent in the classic procedure. The improved repetitive procedure in solution was applied to the preparation of [54-59] fragment of human beta-casein. The structure and high purity of the intermediates, as well as of the final products, was confirmed by FAB-MS, 1H-NMR and HPLC.

Characterisation of selenium compounds in rye seedling biomass using 75Se-labelling/SDS-PAGE separation/gamma-scintillation counting, and HPLC-ICP-MS analysis of a range of enzymatic digests.

In the present study, selenium-enriched plant biomass was investigated to evaluate the ability of rye seedlings to take up, and assimilate, inorganic selenium. Two different analytical approaches were used. Electrophoretic separation (SDS-PAGE) of proteins extracted from 75Se-labelled biomass was used to investigate the biotransformation of selenite into organic forms of the element. Ion-pair chromatography coupled with ICP-MS detection was chosen for the analysis of selenium species, enzymatically extracted from the plant biomass. The results of three enzymatic hydrolysis procedures and three sequential enzymatic extractions procedures are compared. The most effective single extraction was proteolysis (using protease type XIV), giving an overall extraction efficiency of 48%. However, for combinations of enzymes, the most effective was cellulase (Trichoderma viride) followed by sequential extraction of the solid pellet using protease type XIV, giving an extraction efficiency of 70%. The complementary data from the electrophoretic fractionation of proteins, and the HPLC separation of Se-species in the proteolytic digests, reveal the existence of large number of selenium-containing compounds in the rye seedling plant biomass. The results showed the complete biotransformation of inorganic selenium into organic forms during germination of the rye seedlings. HPLC-ICP-MS analysis of extracts from the plant biomass did not show the presence of selenate or selenite. At the time of this study, the lack of suitable organic-MS facilities meant that it was not possible to characterise them fully. However, the data does show that a combination of different enzymes, rather than just the commonly-used protease, should be considered when developing an extraction strategy for selenium in different food types to those already reported in the literature.

Determination of the chlorine kinetic isotope effect on the 4-chlorobenzoyl-CoA dehalogenase-catalyzed nucleophilic aromatic substitution.

The chlorine kinetic isotope effect (KIE) on the dehalogenation of 4-chlorobenzoyl-CoA catalyzed by 4-chlorobenzoyl-CoA dehalogenase has been measured at room temperature and optimal pH. The measured value of (37)k = 1.0090 +/- 0.0006 is larger than the KIEs recently measured for haloalkane and fluoroacetate dehalogenase. This indicates that the transition state for dissociation of chloride ion from the Meisenheimer intermediate is sensitive to the chlorine isotopic substitution. Simple modeling suggests that this sensitivity originates in the high isotopic sensitivity of the C-Cl bond bending modes.

Borderline between E1cB and E2 mechanisms. Chlorine isotope effects in base-promoted elimination Reactions.

The chlorine leaving group isotope effect has been measured for the base-promoted elimination reaction of 1-(2-chloro-2-propyl)indene (1-Cl) in methanol at 30 degrees C: k(35)/k(37) = 1.0086 +/- 0.0007 with methoxide as the base and k(35)/k(37) = 1.0101 +/- 0.0001 with triethylamine (TEA) as the base. These very large chlorine isotope effects combined with large kinetic deuterium isotope effects of 7.1 and 8.4, respectively, are consistent not with the irreversible E1cB mechanism proposed previously (J. Am. Chem. Soc. 1977, 99, 7926) but with the E2 mechanism with transition states having large amounts of hydron transfer and very extensive cleavage of the bond to chlorine.