An Experimental Study on the Visually Recognizable Area

Kuroda, Masami

doi:10.3130/aijsaxx.86.0_28

Cited by 4 publications

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“…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.…”

Section: Methodsmentioning

confidence: 99%

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

Schnackerz

Tai

Pötsch

et al. 1999

Journal of Biological Chemistry

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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...

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Section: Methodsmentioning

confidence: 99%

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

Schnackerz

Tai

Pötsch

et al. 1999

Journal of Biological Chemistry

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“…This topic has been the subject of a recent review (1), and of several more recent publications (2)(3)(4)(5). For every apoenzyme so far studied, analogues of pyridoxal-P with an uncharged or a singly charged anionic group replacing the 5'-phosphate group have been either inactive or have shown only negligible activity as substitutes for pyridoxal-P. A particularly interesting analogue in this connection is pyridoxal 5'-sulfate (6), which has a single strongly acidic ionizable hydrogen on the esterified sulfate group, but is sterically closely similar to pyridoxal 5'-phosphate. The bond angles and distances of phosphate and sulfate are similar (7), the bond lengths differing by only 0.03 A.…”

mentioning

confidence: 99%

Coenzymatic Activity of Pyridoxal 5′-Sulfate and Related Analogues of Pyridoxal 5′-Phosphate

Groman

Huang

Watanabe

et al. 1972

Proc. Natl. Acad. Sci. U.S.A.

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The effect of changes in the substituent at the 5'-position of pyridoxal 5'-phosphate on the coenzymatic activity of six analogues of this coenzyme was determined for three bacterial enzymes. Pyridoxal 5'-sulfate showed substantial coenzymatic activity for arginine decarboxylase and tryptophanase, but not for D-serine dehydratase; a5-pyridoxalmethylphosphonate showed substantial activity for D-serine dehydratase, but not for the other two enzymes. These results demonstrate that neither the dianionic phosphate group nor the ester oxygen of pyridoxal 5'-phosphate is a general requirement for coenzymatic activity. Minor variations in orientation and charge of the group at position 5 exert major effects on the capacity for complex formation between analogue and apoenzyme, and on the catalytic efficiency of the resulting complex, but have comparatively little effect on the substrate affinity of the analogue-apoenzyme complexes. These effects show no general pattern from enzyme to enzyme, and do not correlate with the affinity of analogue for apoenzyme under the conditions tested.

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“…Hi St id!ne Aspartate aminotransferase contains eight histidine residues per monomer (Tu rano £t £|_., 1963;Martinez-Carrion at al., 1967;Banks £l_o, I968;Stankewicz et al«, 1971;Ovchi nni kov et al, 1973). The imidazole group of one of the histidine residues has been shown by various groups of researchers to be of critical importance to the function of the enzyme.…”

Section: Lysinementioning

confidence: 99%

Aspartate aminotransferase: interaction with pyridoxal phosphate analogs and analysis of electronic absorption spectra

Yang

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An Experimental Study on the Visually Recognizable Area

Cited by 4 publications

References 0 publications

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

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

Coenzymatic Activity of Pyridoxal 5′-Sulfate and Related Analogues of Pyridoxal 5′-Phosphate

Aspartate aminotransferase: interaction with pyridoxal phosphate analogs and analysis of electronic absorption spectra

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