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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



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



Biochemistry. 34(6): -1967



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-2apo-beta-2, 790 bar; for alpha-2holo-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 degree C, the corresponding dissociation constants K at 1 bar as well as the reaction volumes of dissociation DELTA-V are calculated as 4.2 times 10-9 M and -196 mL/mol for the apo-beta-2-component and as 9.8 times 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. Pressure release after dissociation of the alpha-2-beta-2 bienzyme complex into native alpha-subunits and completely deactivated beta-protomers leads to 92% recovery of specific beta-2 activity in the spontaneously reassociated apo bienzyme complex (with t-1/2 = 18 min) and to 78% in the holoenzyme (with t-1/2 = 27 min), respectively.

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Accession: 008863653

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PMID: 7849055

DOI: 10.1021/bi00006a017


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