THIAMINASE-I (42-KDA) HETEROGENEITY, SEQUENCE REFINEMENT, AND ACTIVE-SITE LOCATION FROM HIGH-RESOLUTION TANDEM MASS-SPECTROMETRY

Thiaminase I(E.C. 2.5.1.2) from Bacillus thiaminolyticus catalyzes the degradation of thiamin (vitamin B-1). Unexpected mass heterogeneity ( MW 42,127, 42,197, and 42,254; 1:2:1) in recombinant thiaminase I from Escherichia coli was detected by electrospray ionization Fourier-tran sform mass spectrometry, resolving power 7 x 10(4). Nozzle-skimmer fra gmentation data reveal an extra Ala (+ 71.02; 71.04 = theory) and GlyA la (+ 128.04; 128.06 = theory) on the N-terminus, in addition to the f ully processed enzyme. However, the fragment ion masses were consisten t only with this sequence through 330 N-terminal residues; resequencin g of the last 150 bps of the thiaminase I gene yields a sequence consi stent with the molecular weight values and all 61 fragment ion masses. Covalently labeling the active site with a 108-Da pyrimidine moiety v ia mechanism-based inhibition produces a corresponding molecular weigh t increase in all three thiaminase I components, which indicates that they are all enzymatically active. Inspection of the fragment ions tha t do and do not increase by 108 Da indicates that the active site nucl eophile is located between pro(79) and Thr(177) in the 379 amino acid enzyme.