Influence of alpha-subunits on the high-pressure stability of apo and holo beta 2-subunits in the bienzyme complex tryptophan synthase from Escherichia coli.
At a hydrostatic pressure of up to 2 kbar, the isolated alpha-subunit of tryptophan synthase from Escherichia coli proved to be a stable enzyme by virtue of specific activity as well as UV absorption and fluorescence emission spectra. The protein can therefore be regarded as a suitable effector for the investigation of structure-function relationships in the dimeric beta 2-subunit under the influence of high hydrostatic pressure. Complete deactivation of the beta 2-component in the alpha 2 beta 2 bienzyme complex occurs above 1300 bar (midpoint of transition for alpha apo beta 2, 790 bar; for alpha 2 holo beta 2, 1057 bar). Sucrose (13%) shifts both midpoints of transition to values higher by about 300 bar. As shown by sucrose gradient centrifugation and limited trypsinolysis, deactivation of the beta 2-dimer is paralleled by dissociation into denatured beta-chains. At 10 degrees C, the corresponding dissociation constants K at 1 bar as well as the reaction volumes of dissociation delta V are calculated as 4.2 x 10(-9) M and -196 mL/mol for the apo-beta 2-component and as 9.8 x 10(-19) M and -632 mL/mol for the holo-beta 2-component in the bienzyme complex. Furthermore, large negative activation volumes are determined, reflecting the rate increase with increasing pressure: -89 mL/mol for the apo-beta 2-dimer and -195 mL/mol for the holo-beta 2-dimer.(ABSTRACT TRUNCATED AT 250 WORDS)
Institute of Biochemistry, University of Witten/Herdecke, Witten-Annen, FRG.
S
S
Sindern
R
R
van Eldik
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P
Bartholmes
D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D004793:Enzyme Reactivators; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D006874:Hydrostatic Pressure; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014357:Trypsin / Q000378:metabolism; D014367:Tryptophan Synthase / Q000737:chemistry* / Q000378:metabolism
D004793:Enzyme Reactivators; D046911:Macromolecular Substances; D014357:Trypsin; D014367:Tryptophan Synthase