ABSTRACT
Modification of phosphoenolpyruvate carboxylase with o-phthalaldehyde (OPA) resulted in rapid and irreversible inactivation exhibiting biphasic reaction kinetics. The kinetic analysis and correlation of spectral changes with activity indicated that inactivation by OPA results from the modification of two lysine and two cysteine residues per subunit of the enzyme. PEP plus Mg2+ offered substantial protection against modification. Some of the effectors also gave appreciable protection against modification indicating that the residues may be located at or close to the active site. Thus, the results indicate formation of two isoindoles showing the proximity of the essential lysine and cysteine residues at the active site.
Subject(s)
Aldehydes , Binding Sites/physiology , Carboxy-Lyases/metabolism , Kinetics , Phosphoenolpyruvate Carboxylase/antagonists & inhibitors , Zea mays/enzymology , o-PhthalaldehydeABSTRACT
Pyridoxal 5'-phosphate strongly and reversibly inhibited maize leaf 5-amino levulinic acid dehydratase. The inhibition was linearly competitive with respect to the substrate 5-aminolevulinic acid at pH values between 7 to 9·0. Pyridoxal was also effective as an inhibitor of the enzyme but pyridoxamine phosphate was not inhibitory. The results suggest that pyridoxal 5'-phosphate may be interacting with the enzyme either close to or at the 5- aminolevulinic acid binding site. This conclusion was further corroborated by the detection of a Schiff base between the enzyme and the substrate, 5-aminolevulinic acid and by reduction of pyridoxal phosphate and substrate complexes with sodium borohydride.