Limited proteolysis as a structural probe of the soluble alpha-glycerophosphate oxidase from Streptococcus sp.

As reported previously [Parsonage, D., Luba, J., Mallett, T. C., and Claibo rne, A. (1998) J. Biol, Chem, 273, 23812-23822], the flavoprotein cx-glycer ophosphate oxidases (GlpOs) from a number of enterococcal and streptococcal sources contain a conserved 50-52 residue insert that is completely absent in the homologous cl-glycerophosphate dehydrogenases, On limited proteolys is with trypsin, the GlpO from Streptococcus sp. (m = 67.6 kDa) is readily converted to two major fragments corresponding to masses of approximately 4 0 and 23 kDa. The combined application of sequence and mass spectrometric a nalyses demonstrates that the 40-kDa fragment represents the N-terminus of intact GlpO (Met1-Lys368; 40.5 kDa), while the 23-kDa band represents a C-t erminal fragment (Ala405-Lys607; 22.9 kDa). Hence, limited proteolysis in e ffect excises most of the GlpO insert (Ser355-Lys404), indicating that this represents a flexible region on the protein surface. The active-site and o ther spectroscopic properties of the enzyme, including both flavin and tryp tophan fluorescence spectra, titration behavior with both dithionite and su lfite, and preferential binding of the anionic form of the oxidized flavin, were largely unaffected by proteolysis. Enzyme-monitored turnover analyses of the intact and nicked streptococcal GlpOs (at [GlpO] similar to 10 mu M ) demonstrate that the single major catalytic defect in the nicked enzyme c orresponds to a 20-fold increase in K-m(Glp); the basis for this altered ki netic behavior is derived from an 8-fold decrease in the second-order rate constant for reduction of the nicked enzyme, as measured in anaerobic stopp ed-flow experiments. These results indicate that the flexible surface regio n represented by elements of the GlpO insert plays an important role in med iating efficient flavin reduction.