Equilibrium and Kinetic Measurements of the Binding of Pyridoxal 5′‐Phosphate to Hybrid Tryptophan Synthase from Escherichia coli

Abstract

Hybrid tryptophan synthase was prepared that contains a p, subunit with one functional active site while the internal aldimine between cofactor and enzyme of the other protomer was reduced with sodium borohydride (cx,flfl*).The modified enzyme shows a specific activity for the L-serine+indole to L-tryptophan reaction of about 50% compared to the native enzyme [Hathaway, G. M., Kida, S. and Crawford, 1. P., Biochemistry 8, 989-997 (1969)l. The binding of pyridoxal5'-phosphate (pyridoxal-P) to the cx,apopp* complex and to the apoP/?* subunit was studied by equilibrium methods and rapid mixing experiments.The equilibrium binding curves for the hybrid species are hyperbolic (i. e. non-cooperative), yielding apparent microscopic dissociation constants of 1.6kO.2 x M for the holoflp* subunit.The rate of formation of the internal aldimine in the unmodified active site was followed by fast reduction with sodium borohydride, L-tryptophan synthesis was used to monitor the rate of formation of active enzyme.Cofactor binding to the a,apoflP* complex is characterized by three sequential steps of decreasing rate : formation of a non-covalent initial complex, which reacts to an enzymatically inactive internal aldimine, followed by a conformational change leading to the active cx,holoflp* bienzyme complex. A similar series of consecutive steps with decreasing reaction rates is observed for the binding of pyridoxal-P to the apoflP* subunit. The formation of active enzyme, however, is biphasic. The faster process, which parallels the formation of the internal aldimine and which accounts for 8 5 % of the total amplitude, obviously involves an already active high-affinity state of the hybrid molecule whereas the second phase parallels the activation process as observed for cofactor binding to the native dimer.These findings are discussed with respect to the recently proposed mechanism for pyridoxal-P binding to the unmodified apop, subunit [Bartholmes, P. Balk, H. and Kirschner, K, Biochemistry 19, 4527-4533 (1980)l.M for the a,holopfl* complex and 1.5 kO.2 x Tryptophan synthase from Escherichia coli is a multienzyme complex with a z j z structure. It contains aldimine-bound pyridoxal 5'-phosphate (pyridoxal-P) [ 11, which is essential for the reaction catalyzed by the tightly associated dimeric BZ subunit:The protein can be assembled from the isolated c[ and /Iz subunits yielding a complex indistinguishable from the native enzyme. The heterologous interaction between cx and /I2

Circular dichroism studies on the interaction of tryptophan synthase with pyridoxal 5'-phosphate

“…The cofactor itself when in solution or unspecifically bound to other proteins such as bovine serum albumin (Johnson & Graves, 1966) does not show ellipticity in the visible region. Excess unspecific binding of PLP to e-lysyl groups other than the essential one leads to a similar effect (Balk et al, 1981). Comparable observations have been made for denatured PLP-dependent holoenzymes (Fasella & Hammes, 1964a,b).…”

“…At the given wavelength, both apo species reveal no measurable dichroic absorption. Moreover, only the specific interaction of the cofactor with the lysine residue in the active center induces the abovementioned cofactor ellipticity (Balk et al, 1981). Unspecific binding at high concentrations of cofactor (40-fold molar excess) neither influences the specific activity of the enzyme nor gives rise to additional CD effects.…”

“…Buffer. Unless stated otherwise, all experiments were performed in 0.1 M sodium pyrophosphate (pH 7.5), N2 saturated (Balk et al, 1981;Bartholmes et al, 1980). Solutions containing PLP were prepared under yellow light and stored in the dark to prevent photolysis of the cofactor (Reiber, 1972).…”

Limited proteolysis of the .beta.2-dimer of tryptophan synthase yields an enzymatically active derivative that binds .alpha.-subunits

“…The modified /3-chains containing only the larger Nterminal fragment could be purified from the incubation mixture after inhibition of endoproteinase Glu-C with a2macroglobulin. This protein is dimeric and still binds two molecules of pyridoxal 5'-phosphate, however, with hyperbolic characteristics yielding an apparent microscopic dissociation constant Kd of 5 X 10-4 M. This result is similar to the dissociation constant of coenzyme binding to the /3-chain lacking the Goldberg-loop (Kd = 3.4 X 10-4 M, Balk, 1981), which clearly indicates that the integrity of the /3-chain is a necessary prerequisite for the conformational change as taking place upon cooperative pyridoxal 5'-phosphate binding to the native enzyme with Kdl = 8.7 X 10*4 M for the low-affinity T state and K^= 2.3 X 10-7 M for the high-affinity R state (Bartholmes et al, 1980;Balk et al, 1981b).…”