Hideo Kochi

Mechanism of reversible glycine cleavage reaction in Arthrobacter globiformis: Function of lipoic acid in the cleavage and synthesis of glycine

Abstract The hydrogen carrier protein (H-protein) from Arthrobacter globiformis was found to contain one lipoic acid residue per molecule. In the glycine cleavage reaction, H-protein could be replaced by lipoic acid and in glycine synthesis, by dihydrolipoic acid. Lipoamide was less effective than lipoic acid in substituting for H-protein. In glycine synthesis with dihydrolipoic acid, even tetrahydrofolate and T-protein, an enzyme catalyzing the tetrahydrofolate-dependent step of the reaction, were not required, and the whole process of glycine synthesis could proceed in the presence of only one enzyme, P-protein, a pyridoxal phosphate-requiring enzyme. In the glycine decarboxylation reaction with lipoic acid, the decarboxylation product seemed to be bound to lipoic acid, possibly in the ue5f8Sue5f8CH 2 NH 2 form, which was extractable with benzene. The suspected intermediate compound could also be formed nonenzymically from dihydrolipoic acid, formaldehyde and ammonia, and glycine was obtained when the benzene extract containing the suspected intermediate compound was incubated with P-protein and NaHCO 3 . Several lines of evidence suggest that a thiol group of dihydrolipoic acid reacts first with formaldehyde to form a hemimercaptal or mercaptal to which ammonia is subsequently fixed; the binding of ammonia, however, appears to be unstable. Based on observations with free lipoic acids, the sequence of the T-protein-dependent reaction steps in the enzymic, reversible glycine cleavage reaction is discussed.

Reduction of the level of the glycine cleavage system in the rat liver resulting from administration of dipropylacetic acid: An experimental approach to hyperglycinemia☆

Abstract Prolonged administration of dipropylacetic acid, a branched-chain fatty acid, reduced the glycine cleavage activity in the liver of rat to about one-third of the activity in the control rat. The reduction of the activity appeared to be due mainly to reduction of the level of P-protein, a pyridoxal phosphate enzyme, which is responsible for the first step of the glycine cleavage, although dipropylacetic acid was also found to inhibit the activity of P-protein in vitro in a noncompetitive but partially competitive manner with respect to glycine. The rat treated with dipropylacetic acid may provide an experimental approach for the biochemical study of hyperglycinemia which accompanies to metabolic disorders of branchedchain keto acids. In the dipropylacetic acid-treated rat, however, the glycine concentration in the serum was not appreciably elevated and this may be accounted for by the fact that the activities of both the glycine cleavage system and serine dehydratase are considerably higher in the rat liver as compared with those in other animals including human.

Reactions of glycine synthesis and glycine cleavage catalyzed by extracts of Arthrobacter globiformis grown on glycine

Abstract Cell-free extracts of Arthrobacter globiformis grown on glycine as the sole source of of carbon and nitrogen were revealed to catalyze the following reactions: 1. (a) l -Serine + THF ⇌ glycine + methylene-THF, 1 2. (b) l -Serine + CO 2 + NH 3 + 2H ⇌ 2 glycine, 3. (c) Methylene-THF + CO 2 + NH 3 + 2H ⇌ glycine + THF, 4. (d) Glycine + 14 CO 2 ⇌ 14 C-glycine + CO 2 . All these reactions could proceed reversibly. Reaction (d) was strongly suppressed by the addition of THF, reflecting that Reaction (d) is a partial reaction of Reaction (c). Reaction (b) is apparently the sum of Reactions (a) and (c) and provides a new pathway of glycine synthesis from l -serine, CO 2 and ammonia which had been shown to occur in liver mitochondria from various animals. Evidence was obtained that a pyridoxal phosphate enzyme plays a central role in Reactions (c) and (d) and therefore in Reaction (b). Reactions (b), (c), and (d) were also highly dependent upon the presence of thiol compounds such as dithiothreitol and GSH, indicating the involvement of thiol enzyme(s) in these reactions. The glycine cleavage reaction was also strongly stimulated by NAD + , while NADP + had no effect.

Evidence for the presence of a protein-bound intermediate in the cleavage and the synthesis of glycine.

Abstract An intermediate bound to a protein called hydrogen carrier protein was isolated from an incubation of the glycine metabolizing system with glycine or methylene tetrahydrofolate followed by filtration on Sephadex G-100. The possible mechanism of the metabolism of glycine was discussed.