Interactions between Tryptophan Synthase from Escherichia coli and Derivatives of the Coenzyme Pyridoxal 5'-Phosphate

Abstract

Abstract 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.