Thiamin phosphate synthase catalyzes the coupling of 4-methyl-5-(beta-hydroxyethyl)thiazole phosphate (Thz-P) and 4-amino-5-(hydroxymethyl)-2-methylpyrimidine pyrophosphate (HMP-PP) to give thiamin phosphate. In this paper, we demonstrate that 4-amino-5-(hydroxymethyl)-2-(trifluoromethyl)pyrimidine pyrophosphate (CF(3)-HMP-PP) is a very poor substrate [k(cat)(CH(3)) > 7800k(cat)(CF(3))] and that 4-amino-5-(hydroxymethyl)-2-methoxypyrimidine pyrophosphate (CH(3)O-HMP-PP) is a good substrate [k(cat)(OCH(3)) > 2.8k(cat)(CH(3))] for the enzyme. We also demonstrate that the enzyme catalyzes positional isotope exchange. These observations are consistent with a dissociative mechanism (S(N)1 like) for thiamin phosphate synthase in which the pyrimidine pyrophosphate dissociates to give a reactive pyrimidine intermediate which is then trapped by the thiazole moiety.

A widely used procedure for site localization of covalent protein modifications involves proteolysis, partial chromatographic separation of the resulting complex mixture, and tandem mass spectrometry (MS/MS) to identify peptides whose molecular weight (M r ) has been increased appropriately by the modification. As found previously for MS of small molecules, this study shows that protein fragment identification can be greatly simplified by labeling the modification with stable isotopes. Further, the high resolution capabilities of Fourier transform MS make possible the direct identification of CH 3 /CD 3 -labeled peptides without chromatographic separation. Although separate Asp-N, Lys-C, and ␣-chy- Biological activity can be dramatically altered by a small covalent modification in molecular structure (1, 2). To characterize such modifications in small molecules, the sensitivity, speed, and specificity of tandem mass spectrometry (MS/MS) 1 (3-5) provide well recognized advantages over other methods (e.g. Edman, NMR, IR, crystallography) (2, 6, 7). For example, identifying drug metabolites in a complex extract is especially straightforward if a methyl group of the drug molecule has been partially replaced by CD 3 ; even if the mass spectrum contains many molecular ions, any showing isotopic peaks separated by 3 Da are probably those of a metabolite (3,8,9). Further, MS/MS isolation and fragmentation of each isotopically unique ion group can provide information on the label's location in the molecule. Characterizing post-translational or other covalent modifications in large biomolecules is even more challenging (6); the most effective MS methods (4, 10, 11) for proteins of known sequence (amounts as low as 3 ϫ 10 Ϫ14 mol) (10) employ extensive proteolysis, chromatographic fractionation, and MS/MS of the peptide fractions. The masses of the peptides originating from the protein without and with the label can be compared to identify the labeled peptide, followed by its characterization by MS/MS, or all of the labeled protein's peptides can be examined by MS/MS to identify and characterize the labeled peptide. Here this examination is simplified with mixed, preferably isotopically mixed, labels. Further, the chromatographic steps are eliminated by utilizing the Ͼ10 5 MS/MS resolving power of Fourier transform (FT) MS (5,12,40). This is illustrated with identification of the active-site nucleophile of thiaminase I, a 379-amino acid enzyme. Illustrating the potential unique applicability of this methodology, MS/MS data sufficient for identification in the Protein Data Bank (13) have been obtained by ESI/FTMS recently from 10 Ϫ17 mol of a 29-kDa protein (14). Thiaminase I from Bacillus thiaminolyticus catalyzes the degradation of thiamin (vitamin B 1 ; Scheme I, 1) by nucleophilic displacement of its thiazole moiety (15,17,41,42). Treatment with the suicide substrate 4-amino-6-chloro-2-methylpyrimidine (2) caused the molecular ion mass to increase by the 107 Da expected for a single adduct (16). Fragmentation of these m...

No abstract

No abstract

No abstract