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

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.

Substitution of Pyridoxal 5′-Phosphate in the O-Acetylserine Sulfhydrylase from Salmonella typhimurium by Cofactor Analogs Provides a Test of the Mechanism Proposed for Formation of the α-Aminoacrylate Intermediate

“…Replacement of the native PLP cofactor with analogs has been used in other systems to map the cofactor site and obtain additional information on the reaction mechanism (11)(12)(13)(14)(15)(16)(17)(18). Recently the resolution of the PLP cofactor from OASS was achieved by dissociation of the ␣-aminoacrylate intermediate from the active site in the presence of 5 M guanidinium chloride (18).…”