The effect of pyridoxal phosphate on rabbit muscle aldolase

Abstract

Rabbit muscle aldolase loses activity when exposed to low concentrations of pyridoxal 5′-phosphate. The inactivation is specifically prevented by substrates and substrate analogues, and is reversed by dilution, or by addition of amino thiols. Pyridoxal 5′-phosphate reacts with approximately six ϵ-amino lysine groups in the enzyme. The product was identified by spectral studies and by reduction with NaBH 4 followed by hydrolysis and isolation of N 6-pyridoxyllysine. Peptides containing N 6-pyridoxyllysine were obtained after cyanogen bromide cleavage of the reduced complex, and the lysine residues that reacted with PLP were shown to be distinct from those involved in Schiff base formation with the substrate. The evidence suggests that pyridoxal 5′-phosphate reacts at one of the phosphate-binding sites of rabbit muscle aldolase. Most of the bound pyridoxal phosphate was located in the amino-terminal peptide, which contained one equivalent of pyridoxyl-P per subunit. Smaller quantities, about one-half equivalent per subunit, were incorporated into the carboxyl-terminal peptide. The lysine residue that forms the Schiff base with dihydroxyacetone phosphate was present in a third peptide located near the center of the chain.