Chemical Modification of Pseudomonas ochraceae 4-Hydroxy-4-Methyl-2-0xoglutarate Aldolase by Diethyl Pyrocarbonate

Abstract

Diethyl pyrocarbonate inactivates Pseudomonas ochraceae 4-hydroxy-4-methyl-2-oxo-glutarate aldolase [4-hydroxy-4-methyl-2-oxoglutarate pyruvate-lyase: EC 4.1.3.17] by a simple bimolecular reaction. The inactivation is not reversed by hydroxylamine. The pH curve of inactivation indicates the involvement of a residue with a p K of 8.8. Several lines of evidence show that the inactivation is due to the modification of ε-amino groups of lysyl residues. Although histidyl residue is also modified, this is not directly correlated to the inactivation. No cysteinyl, tyrosyl, or tryptophyl residue or α-amino group is significantly modified. The modification of three lysyl residues per enzyme subunit results in the complete loss of aldolase activity toward various 4-hydroxy-2-oxo acid substrates, whereas oxaloacetate β-decarboxylase activity associated with the enzyme is not inhibited by this modification. Statistical analysis suggests that only one of the three lysyl residues is essential for activity. l-4-Carboxy-4-hydroxy-2-oxoadipate, a physiological substrate for the enzyme, strongly protects the enzyme against inactivation. P_l as an activator of the enzyme shows no specific protection. The molecular weight of the enzyme, Km for substrate or Mg²⁺, and activation constant for P_l are virtually unaltered after modification. These results suggest that the modification occurs at or near the active site and that the essential lysyl residue is involved in interaction with the hydroxyl group but not with the oxal group of the substrate.