PMID: 701292Nov 10, 1978Paper

Active site studies of ribulose-1,5-bisphosphate carboxylase/oxygenase with pyridoxal 5'-phosphate

The Journal of Biological Chemistry
C Paech, N E Tolbert

Abstract

There are 16 epsilon-amino groups of lysyl residues which are essential for the activity of ribulose-1,5-bisphosphate carboxylase/oxygenase. These lysyl residues formed a Schiff base with pyridoxal 5'-phosphate which was stabilized by NaBH4 reduction. The stoichiometry of covalently bound pyridoxal 5'-phosphate after NaBH4 reduction was determined spectrophotometrically with a derived molar extinction coefficient of 4800 M-1 cm-1. The incorporation of pyridoxal 5'-phosphate into the protein was accompanied by loss of the carboxylase and oxygenase activities, but the ratio of their activities remained constant. Vmax, but not Km, values were changed by this modification of the amino acid groups. Half of the epsilon-amino groups of lysine appeared to be at the 8 catalytic sites and half at the 8 activator sites for CO2, as indicated by kinetics of reactivation of the enzyme activity during dissociation of the Schiff base between pyridoxal 5'-phosphate and the protein. Reduction with NaB3H4 revealed that all 16 of the lysyl residues were on the large subunit. Ribulose-1,5-bis-phosphate alone protected 16 amino groups from Schiff base formation with pyridoxal 5'-phosphate, and the enzyme activity was fully conserved. NaHCO3 increase...Continue Reading

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