The mechanism of pryidoxal 5'-phosphate (PLP) binding to both the alpha apo beta 2 complex and the apo beta 2 subunit of tryptophan synthase was investigated by rapid mixing experiments. Absorption and fluorescence changes were used to monitor the binding reaction directly. Reduction with sodium borohydride provided the rate of formation of the internal aldimine with the lysine amino group of the enzyme, and substrate turnover monitored the rate of formation of active enzyme. The alpha 2 apo beta 2 complex binds PLP in a sequence of three steps of decreasing rate: formation of a noncovalent complex, which isomerizes to an enzymically inactive internal aldimine, followed by formation of an active alpha 2 holo beta 2 complex. The two binding sites appear to bind PLP independently. The apo beta 2 subunit binds PLP cooperatively in a sequence of three steps of decreasing rate: formation of a noncovalent complex, which isomerizes to an enzymically inactive internal aldimine, followed by the formation of the enzymically active holo beta 2 subunit. Taken together with kinetic studies of pyridoxine phosphate binding [Tschopp, J., & Kirschner, K. (1980) Biochemistry (second paper of three in this issue)], the rate data of the apo beta 2 subunit are shown to be consistent with the concerted mechanism. The difference between the values of the isomerization rate constants of bound PLP and bound PNP appear to result from the covalent internal aldimine, which is formed with PLP but not with PNP.

The interaction between the beta 2 subunit of tryptophan synthase and the coenzyme pyridoxal 5'-phosphate (PLP) is characterized by induced circular dichroism (CD) in the near-UV (260-285 nm) and in the visible region (320-480 nm, extrinsic Cotton effect). Because of its high mean residue ellipticity ([theta] = 56 deg cm2 dmol-1 for the isolated holo-beta 2 subunit and 102 deg cm2 dmol-1 for the alpha 2-holo-beta 2 complex, respectively) the latter has been used to define different conformational states of the beta 2 dimer via CD titrations. Fitting the obtained binding parameters to the known data from equilibrium dialysis leads to the result that the low-affinity state of the isolated beta 2 subunit shows a 3 times greater rotational strength than the holoenzyme in the high-affinity state. The generation of the final CD amplitude is characterized by a rate constant intermediate between the values for the formation of the internal aldimine and for the regain of enzymatic activity. Interaction of the alpha 2-apo-beta 2 bienzyme complex with the cofactor leads to a hyperbolic binding curve which is apparently free of contributions caused by unspecific PLP binding outside the active center. The determined dissociation constant of 9 x 10(-7) M is in good agreement with the value of 1 x 10(-6) M as obtained by equilibrium dialysis. Binding kinetics reveal a very slow process with a rate constant of 2.6 x 10(-4) s-1, significantly smaller than that for the regain of catalytic activity during reconstitution of the enzyme.

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

The interaction of the coenzyme analogues pyridoxal (A), pyridoxine 5'-phosphate (B), pyridoxic acid 5'-phosphate (Q and N-phosphopyridoxyl-L-serine (D) with both the isolated apo β2 subunit and the native < x2 apo β2 bienzyme complex of tryptophan synthase from Escherichia coli has been investigated using enzyme kinetics and CD spectroscopy. A 500-fold molar excess of (A) yields a maximum activation of the a2 apo β2 complex of 12% compared to the native holo bienzyme complex. The corresponding Michaelis constant KM equals 0.16 mM. Compounds (B -D) which lack the reactive carbonyl group in the 4-position cannot act as cofactor during enzymic turnover. However, they are competitive inhibitors with respect to the natural coenzyme pyridoxal 5'-phosphate. The corresponding inhibition constants K\ are for (B): 0.10 mM, (C): 0.03 mM and (D): 0.16 mM.The CD spectra of the aromatic side chains of both protein species ([@]278nm for the β2 subunit = 26 degr cm2 decimol-1, for the bienzyme complex = 40 degr cm2 decimol-1) remain un­ changed and no measurable dichroic absorption is induced in the visible region at 415 nm upon addition of (A), (at this wavelength productive binding of pyridoxal 5'-phosphate induces a significant extrinsic Cotton effect in the internal aldimine). Reaction with (B) leads to an enhancement of the dichroic amplitude at 278 nm of the isolated β2 subunit {A[6] = 6 degr cm2 decimol-1) and of the a2 β2 complex (zl[0] = 17 degr cm2 decimol-1) respectively. Com­ pound (C) shows no effect in the aromatic region of the β2 subunit, but a decrease in the a2 β2 complex (A[0) = 5 degr cm2 decimol-1). At 315 nm, however, a remarkable extrinsic Cotton effect of + 20 degr cm2 decimol-1 is induced in (Q . Ligand (D) causes a similar increase at 278 nm of A[0] = 14 degr cm2 decimol-1 in both protein species. The given data are discussed on the basis of the mode of binding of the natural coenzyme.

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