Microcrystals of tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium are catalytically active.
An improved and efficient method has been developed for the purification of the tryptophan synthase alpha 2 beta 2 complex (EC 4.2.1.20) from Salmonella typhimurium containing a multicopy plasmid. Microcrystals prepared in 12% poly(ethylene glycol) 8000 containing 2.5 mM spermine are shown by scanning electron microscopy to have the same crystal habit as the larger crystals that are being used for structural analysis by X-ray crystallography. The average dimensions of the crystals are 33 microns (length) X 9 microns (width) X 3 microns (maximum thickness). Our finding that suspensions of microcrystals are active in several reactions catalyzed by the active sites of the alpha and beta 2 subunits demonstrates that both active sites are functional in the crystal and accessible to substrates. Thus the larger crystals being used for X-ray crystallographic studies should form complexes with substrates and analogues at both active sites and should yield functionally relevant structural information. A comparison of the reaction rates of suspensions of microcrystals with those of the soluble enzyme shows that the maximum rate of the crystalline enzyme is 0.8 that of the soluble enzyme in the cleavage of indole-3-glycerol phosphate (alpha reaction), 0.3 that of the soluble enzyme in the synthesis of L-tryptophan by the beta reaction or the coupled alpha beta reaction, and 2.7 that of the soluble enzyme in the serine deaminase reaction. These small differences in rates probably reflect functional differences between the crystalline and soluble enzymes since the reaction rates of the microcrystals are calculated to be virtually free of diffusional limitation under these reaction conditions.(ABSTRACT TRUNCATED AT 250 WORDS)
Ahmed
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SA
Laboratory of Biochemical Pharmacology, National Institute of Diabetes, Bethesda, Maryland 20892.
Hyde
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CC
Thomas
G
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Miles
E W
EW
D002852:Chromatography, Ion Exchange; D003460:Crystallization; D007700:Kinetics; D046911:Macromolecular Substances; D008855:Microscopy, Electron, Scanning; D012486:Salmonella typhimurium / Q000201:enzymology*; D014367:Tryptophan Synthase / Q000302:isolation & purification* / Q000378:metabolism
D046911:Macromolecular Substances; D014367:Tryptophan Synthase