Substitution of Pyridoxal 5′-Phosphate ind-Serine Dehydratase from Escherichia coliby Cofactor Analogues Provides Information on Cofactor Binding and Catalysis

Abstract

A second low affinity binding site for the cofactor has also been reportedFrom reconstitution experiments of D-serine apodehydratase with various PLP analogues, it was deduced that substitutions at positions 2 and 6 of the coenzyme are not critical for catalytic activity of the dehydratase (3). However, the phosphate group in the 5Ј-position of the cofactor in its dianionic form is essential (4, 5). The absorption spectrum of highly purified DSD exhibits a prominent absorbance maximum at 415 nm, indicating a protonated Schiff base linkage of the formyl group of PLP to the ⑀-amino group of Lys-118 (1, 6, 7). Excitation of D-serine dehydratase at 296 nm causes an emission spectrum with maxima at 335 and 510 nm, respectively (8 -11). The emission maximum at 510 nm is due to energy transfer between a tryptophanyl residue and PLP (9). The dehydratase is weakly fluorescent with an excitation maximum at 415 nm corresponding to the absorbance maximum of the internal Schiff base and an emission maximum around 510 nm (8 -11), typical of all PLP-dependent enzymes studied so far (12-16). Upon addition of 0.5 M glycine or L-alanine the fluorescence intensity at 510 nm increased markedly, and the max shifts from 510 to 485 (9, 10), similar to spectral changes observed for O-acetylserine sulfhydrylase (12). As suggested above a particularly useful approach to investigate the active sites of PLP enzymes is measuring spectral properties associated with the protein and cofactor. In this study we have investigated the environment of the natural cofactor and three cofactor analogues, one active and two inactive, in DSD by CD and fluorescence spectroscopy. EXPERIMENTAL PROCEDURESChemicals-PDMP was kindly provided by Dr. O. Saiko (Merck). PLP monomethyl ester, prepared by the method of Pfeuffer et al. (17), was a gift of Dr. J. Ehrlich. Pyridoxal 5Ј-sulfate was prepared by the method of Kuroda (18). All other chemicals were of highest quality commercially available.Enzymes-D-Serine dehydratase was purified from Escherichia coli K12 mutant C6 as described by Schiltz and Schnackerz (19) or from a wild-type DSD expression plasmid (11). D-Serine apodehydratase was prepared by using the resolution procedure described by Dowhan and Snell (1). The apoenzyme had a residual activity of 1.9 units/mg of protein. The specific activity of PLP-reconstituted dehydratase was 100 units/mg of protein when measured at 25°C. Reconstitution of apodehydratase with PLP analogues was achieved by incubating apoenzyme with a 5-fold excess of the respective cofactor analogue for 1 h at 25°C in the dark. Excess cofactor analogue was removed by passing the incubation mixture over a Sephadex G-25 (3.4 ϫ 40 cm) or PD10 column equilibrated with 100 mM potassium phosphate buffer, pH 7.8. The specific activity of the PDMP-reconstituted dehydratase was 38 units/mg of protein.Enzyme Assay-The enzymatic activity of DSD was determined at 25°C as described by Dowhan and Snell (1). The assay mixture contains * This work was supported in part by funds from the Deutsche For...

Spectral and kinetic characterization of 7,8-diaminopelargonic acid synthase from Mycobacterium tuberculosis

“…3B), the absorption maxima, indicates the presence of a positive induced cotton effect, a manifestation of the specific structure of the PLP-enzyme complex. PLP, being achiral is not intrinsically optically active but is induced to be so by the asymmetric binding environment of the active site (21). The transition of the positive cotton effect peak from 420 nm to 335 nm is an indication of the change in the torsion angle of the C4-C4 0 bond, a major determinant of the cotton effect (26).…”

“…The presence of the cofactor endows PLP-dependent enzymes with a unique spectral signature which is sensitive to changes in protein structure and/or the orientation of the cofactor (21). It is, therefore, a useful means for comparing PLP-dependent enzymes, which are known to catalyze reactions as diverse as transamination, racemization, decarboxylation, b-elimation and retro-aldol cleavage (22).…”

“…CD spectra are useful in interpreting overall structural parameters of a protein and the orientation of the cofactor (21). The presence of the band centered on 420 nm (Fig.…”

“…Information regarding the presence of various tautomers of the protein-bound PLP is provided by UV-visible absorption spectra (21). The two absorption bands, one at 420 nm and another at 335 nm seen in the UV-visible absorption spectrum of DAPA synthase, represent the ketoenamine and enolimine tautomers, respectively, of the protonated internal aldimine, formed between the cofactor PLP and the e-amino group of Lysine.…”

“…Fluorescence emission spectra generally provide information about the environment and orientation of the cofactor in PLP-dependent enzymes (21). The emission maximum at 338 nm obtained for DAPA synthase upon excitation at 280 nm (Fig.…”