Subunit interaction in tryptophan synthase of Escherichia coli: calorimetric studies on association of .alpha. and .beta.2 subunits

Abstract

Association of the apo-.beta.2 and the holo-(.beta.-PLP)2 subunits of tryptophan synthase from E. coli (L-serine hydro-lyase (adding indole) (EC 4.2.1.20)) with .alpha. subunits of the same enzyme was studied by microcalorimetry. The results obtained from thermometric titrations clearly demonstrate that only the native complex .alpha.2.beta.2 is formed, independent of an excess of .alpha. protein. The reaction of the holo-(.beta.-PLP)2 with .alpha. subunits at 25 C is accompanied by a negative enthalpy change, which is almost twice as large as that for complex formation with the apo-.beta.2 protein, thus indicating that the interaction enthalpy becomes more favorable in the presence of the coenzyme pyridoxal 5''-phosphate (PLP). Both reaction enthalpies show very large negative temperature coefficients, -3600 .+-. 100 cal [calories] K-1 (mol of .beta.2)-1 being the value for the formation of the apoenzyme and -2300 .+-. 100 cal.degree. K-1 (mol of .beta.2)-1 pertaining for formation of the holoenzyme. The studies on the association of .alpha. and .beta.2 subunits in the 2 buffers revealed that at 25.degree. C, approximately 0.75 proton are absorbed in the presence and absence of the coenzyme, whereas at 35.degree. C 1 proton is taken up from the solution when PLP is present, but 2 if the apo-.beta.2 complex reacts. These results are a clear indication of energetic linkage between intersubunit interaction, H+ equilibria, and the binding of the coenzyme.