COENZYMATIC ACTIVITIES OF 2-NOR-2-HYDROXY-METHYL PYRIDOXAL 5'-PHOSPHATE FOR VARIOUS VITAMIN B6 ENZYMES

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概要

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• The coenzymatic activities of 2-nor-2-hydroxymethyl-pyridoxal 5'-phosphate (2'-hydroxy PLP) for various vitamin B6-dependent enzymes were studied in order to obtain information on the role of the methyl group at position 2 of pyridoxal 5'-phosphate (PLP) for the binding to apoprotein and for the catalytic action of the holoenzyme. Although it has been demonstrated that this coenzyme analog binds to the catalytic center and serves as a coenzyme for a variety of B6 en-zymes, the mode of binding as well as the catalytic activity of the holo-enzyme reconstituted with the analog was different according to the kinds of enzyme and assay conditions. In the ordinary assay system using Tris-HCl buffer, 2'-hydroxy PLP showed almost an equal affinity for pig heart muscle mitochondrial and cytoplasmic aspartate aminotransferase (GOTs, and GOTm) to that of PLP. The maximal velocity (Vmax value) of the reaction mediated by GOTs, or GOTm reconstituted with 2'-hydroxy PLP was somewhat large as compared with that catalyzed by the native holoenzyme. How-ever, when phosphate buffer was used for the assay system, the affinity of the coenzyme analog decreased markedly and the Vmax value catalyzed by the analog-bound GOTs, or GOTm became approximately 70% of that by the native enzyme. Thus as judged from the Vmax value under ordinary assay conditions, 2'-hydroxy PLP is considered to be rather superior to PLP as the coenzyme for GOT. Phosphate anion inhibited the binding of PLP or 2'-hydroxy PLP to apoGOT in a competitive manner and further caused an appreciable decrease in the apparent Vmax of the reaction catalyzed by the resulting holoenzyme. The K1, values of phosphate anion against PLP and 2'-hydroxy PLP were 11.9 and 3.75, respectively. On the other hand, in the case of E. coli tryptophanase, both the affinity for 2'-hydroxy PLP and the Vmax value of the reaction mediated by the analog-bound enzyme were significantly smaller than those of the native coenzyme irrespective of the buffer used. Of the other B6 enzymes tested hitherto, E. intermedia tyrosine phenol-lyase and Pseudomonas dacunhae aspartate β-decarboxylase showed the same behaviors toward 2'-hydroxy PLP as that of tryptophanase. On the contrary, the coenzyme analog served as a more efficient co-enzyme than the native coenzyme for E. coli D-serine dehydrase in the presence of phosphate anion. These results indicate that there are at least three groups of B6 enzymes exhibiting different interaction with the 2-methyl group of PLP.

• 財団法人 学会誌刊行センターの論文

著者

• 清水 祥一

  Laboratory of Industrial Biochemistry, Department of Industrial Chemistry, Faculty of Engineering, Kyoto University

• 真杉 文紀

  Laboratory of Industrial Biochemistry, Department of Industrial Chemistry, Faculty of Engineering, Kyoto University

• 名取 与平

  Laboratory of Industrial Biochemistry, Department of Industrial Chemistry, Faculty of Engineering, Kyoto University

• 福井 三郎

  Laboratory of Industrial Biochemistry, Deparmtnt of Industrial Chemistry, Faculty of Engineering, Kyoto University

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• INHIBITION OF ACTIVITIES OF ASPARTATE AMINO-TRANSFERASE AND TRYPTOPHANASE BY EXCESS BINDING OF PYRIDOXAL 5'-PHOSPHATE
• COENZYMATIC ACTIVITIES OF 2-NOR-2-HYDROXY-METHYL PYRIDOXAL 5'-PHOSPHATE FOR VARIOUS VITAMIN B6 ENZYMES
• TRANSFER OF PYRIDOXAL 5'-PHOSPHATE FROM ALBUMIN-PYRIDOXAL 5'-PHOSPHATE COMPLEX TO APO-ASPARTATE AMINOTRANSFERASE
• Spectrophotometric Demonstration of Vitamin B12S-Formation by Nucleophilic Cleavage of Co-C Bond of Acetylcobalamin
• STUDIES ON THE OXIDATION PRODUCTS OF LIPOIC ACID

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