Conversion of glycerate to serine in intact spinach leaf peroxisomes

Abstract

Intact spinach ( Spinacia oleracea L.) leaf peroxisomes converted glycerate to serine in the presence of NAD and alanine. The reaction proceeded optimally at pH 9. Addition of oxaloacetate or α-ketoglutarate plus aspartate enhanced the conversion about three-fold. Alteration of the concentration of one of the reaction components, consisting of 2 m m glycerate, 0.2 m m NAD, 0.5 m m oxaloacetate, and 2 m m alanine, revealed half-saturation constants of 0.45 m m for glycerate, 0.06 m m for NAD, 0.02 m m for oxaloacetate, and 0.33 m m for alanine. The conversion proceeded with the formation of hydroxypyruvate followed by serine; hydroxypyruvate did not accumulate to a high amount in the presence or absence of alanine. The amino group donor could be alanine (half-saturation constant, 0.33 m m), glycine (0.45 m m), or asparagine (0.67 m m); the three amino acids produced roughly similar V max values. The results indicate that, in the conversion of glycerate to serine, the transamination is catalyzed by a hydroxypyruvate aminotransferase with characteristics unknown among all other studied leaf peroxisomal aminotransferases. The peroxisomal membrane is sparsely permeable to NAD/NADH, and the participation of the peroxisomal malate dehydrogenase in an electron shuttle system across the membrane in the regeneration of NAD/NADH is suggested.