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  • Title: Substrate-induced changes in sulfhydryl reactivity of bacterial D-amino acid transaminase.
    Author: Soper TS, Ueno H, Manning JM.
    Journal: Arch Biochem Biophys; 1985 Jul; 240(1):1-8. PubMed ID: 4015092.
    Abstract:
    D-Amino acid transaminase from Bacillus sphaericus strain ATCC 14577 is a dimer with eight cysteinyl residues per molecule (T.S. Soper, W.M. Jones, and J.M. Manning (1979) J. Biol. Chem. 254, 10,901-10,905). The reaction of the cysteinyl residues with a variety of sulfhydryl reagents has been explored to gain insight into the physical environments around these cysteinyl residues in the absence or the presence of substrates. The native enzyme, in the pyridoxal-P conformation, appears to be a symmetrical dimer, whose SH groups react in pairs with anionic reagents such as 5,5'-dithiobis(2-nitrobenzoic acid) or the halo acids. Two SH groups react with either reagent without altering enzymatic activity. Two additional SH groups react with DTNB with loss of catalytic activity. Positively charged reagents such as beta-bromoethylamine are much more effective in inactivating the pyridoxal-P conformation of the enzyme with almost five of the eight SH groups reacting and this results in a significant loss in catalytic activity. The neutral reagent dithiodipyridine is able to detect some asymmetry in the pyridoxal-P conformation. Upon addition of a D-amino acid substrate, the enzyme is transformed into the pyridoxamine-P conformation. This conformation is much more reactive with anionic reagents and much less reactive with cationic reagents, suggesting that there is a significant change in the net charge around one of the SH groups in the pyridoxamine-P conformation. Also, titration with DTNB indicates that the enzyme is a much more asymmetric dimmer in the pyridoxamine-P conformation than in the pyridoxal-P conformation. Thus, upon binding of a D-amino acid substrate, D-amino acid transaminase is transformed into the pyridoxamine-P conformation. This results in a significant change in the environment of four of the sulfhydryl groups of the enzyme. We conclude that the enzyme is transformed from a symmetrical dimer into an asymmetrical dimer and that the net charge of one of the pairs of cysteinyl groups is changed from a net negative charge into a net positive charge. These results suggest that there is a significant conformational change that occurs during the transition from the pyridoxal-P into the pyridoxamine-P form of this transaminase.
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