INFLUENCE OF ALPHA-SUBUNITS ON THE HIGH-PRESSURE STABILITY OF APO ANDHOLO 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 enz yme by virtue of specific activity as well as UV absorption and fluore scence emission spectra. The protein can therefore be regarded as a su itable effector for the investigation of structure-function relationsh ips in the dimeric beta(2)-subunit under the influence of high hydrost atic pressure. Complete deactivation of the beta(2)-component in the a lpha(2) beta(2) bienzyme complex occurs above 1300 bar (midpoint of tr ansition for alpha(2)apo beta(2), 790 bar; for alpha(2)holo beta(2), 1 057 bar). Sucrose (13%) shifts both midpoints of transition to values higher by about 300 bar. As shown by sucrose gradient centrifugation a nd limited trypsinolysis, deactivation of the beta(2)-dimer is paralle led by dissociation into denatured beta-chains. At 10 degrees C, the c orresponding 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. Fur thermore, large negative activation volumes are determined reflecting the rate increase with increasing pressure: -89 mL/mol for the apo-bet a(2)-dimer and -195 mL/mol for the holo-beta(2)-dimer. Pressure releas e after dissociation of the alpha(2) beta(2) bienzyme complex into nat ive alpha-subunits and completely deactivated beta-protomers leads to 92% recovery of specific beta(2) activity in the spontaneously reassoc iated apo bienzyme complex (with t(1/2) = 18 min) and to 78% in the ho loenzyme (with t(1/2) = 27 min), respectively.