Application of microcolumn liquid chromatography-continuous-flow fast atom bombardment mass spectrometry in environmental studies of sulfonylurea herbicides.

The use of 0.25-mm I.D. packed capillary liquid chromatography columns coupled with continuous-flow fast atom bombardment (FAB) mass spectrometry has proven to be a very valuable technique, especially for the identification of unknown sulfonylurea herbicide metabolites. Several new and unusual heterocycle ring-opened metabolites and hydrolysis products were identified, and metabolic pathways were proposed. Typical column flow-rates are 1-2 microliters/min, which allows direct coupling with no sample splitting. This is important in our metabolite identification work, since we are usually sample-limited. Techniques for increasing injection volume to allow analyses of dilute solutions and the use of polymeric packing for separation of polar metabolites are discussed. The FAB mass spectra usually provide unequivocal molecular weights and structurally useful fragments ions, which often allows structure assignments on exceedingly small quantities of isolated metabolites.

The mechanism of phenylalanine hydroxylase.

The site of oxygen binding during phenylalanine hydroxylase (PAH)-catalyzed turnover of phenylalanine to tyrosine has been tentatively identified as the 4a position of the tetrahydropterin cofactor, based on the spectral characteristics of an intermediate generated from both 6-methyltetrahydropterin and tetrahydrobiopterin during turnover. The rates of appearance of the intermediate and tyrosine are equal. Both rates exhibit the same dependence on enzyme concentration. PAH also requires 1.0 iron per 50,000-dalton subunit for maximal activity. A direct correlation between iron content and specific activity has been demonstrated. Apoenzyme can be reactivated by addition of Fe(II) aerobically or Fe(III) anaerobically and can be repurified to give apparently native protein. Evidence from electron paramagnetic resonance implicates the presence of high spin (5/2) Fe(III). As a working hypothesis we postulate that a key complex at the active site may be one containing iron in close proximity to a 4a-peroxytetrahydropterin.

Phenylalanine hydroxylase. Correlation of the iron content with activity and the preparation and reconstitution of the apoenzyme.

Phenylalanine hydroxylase requires 1.0 mol of iron/Mr = 50,000 subunit for maximal activity. A direct correlation between iron content and specific activity has been demonstrated through a comparison of enzyme activity and iron bound per subunit for various enzyme preparations and a measurement of the remaining activity upon partial and total removal of the iron by chelation. Apoenzyme has been prepared that can be reactivated by addition of Fe(II) aerobically or of Fe(III) anaerobically. A comparison of the native and reconstituted phenylalanine hydroxylase shows the latter behaves identically upon affinity and Chelex column chromatography as well as precipitation with ammonium sulfate supporting its close similarity to the native enzyme.

On the mechanism of action of phenylalanine hydroxylase.

The oxidation of 6-methyltetrahydropterin and tetrahydrobiopterin coupled to the formation of tyrosine by phenylalanine hydroxylase generates a precursor species to the quinonoid product that is tentatively identified as a 4a-hydroxy adduct based on its spectral similarity to the 4a-hydroxy-6-methyl-5-deazatetrahydropterin. The rate of appearance of this intermediate and that of tyrosine are equal and hydroxylase catalyzed in accord with the completion of the hydroxylation event. This observation, which confirms and extends an earlier one by Kaufman [Kaufman, S. (1975) in Chemistry and Biology of Pteridines (Pfleiderer, W., Ed.) p 291, Walter de Gruyter, Berlin], serves to link the reaction courses followed by pterin and pyrimidine cofactor analogues and supports the hypothesis that the 4a position is a site of O2 attachment. Thus, as expected, no prereduction of the enzyme was observed in anaerobic experiments utilizing stoichiometric amounts of enzyme and tetrahydropterin in the presence or absence of 1 mM phenylalanine. Activation of the hydroxylase by 1 mM lysolecithin leads to oxidation of the tetrahydropterin in the absence of phenylalanine. A ring-opened pyrimidine analogue of the tetrahydropterin, 2,5-diamino-4-[(meso-1-methyl-2-aminopropyl)amino]-6-hydroxypyrimidine, was studied to examine the possibility of tetrahydropterin ring opening in the enzymatic reaction prior to 4a-hydroxy adduct formation. However, no hydroxylase-catalyzed ring closure was observed.