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Formaldehyde Binding by 2-Keto-4-hydroxyglutarate Aldolase

FORMATION AND CHARACTERIZATION OF AN INACTIVE ALDOLASE-FORMALDEHYDE-CYANIDE ADDUCT

From the ¹ From the Department of Biological Chemistry, The University of Michigan, Ann Arbor, Michigan 48104

2-Keto-4-hydroxyglutarate aldolase (2-oxo-4-hydroxyglutarate glyoxylate-lyase:2-oxo-4-hydroxyglutarate pyruvate + glyoxylate), which catalyzes a terminal step in the catabolism of l-hydroxyproline by mammals, selectively interacts with formaldehyde; a Schiff-base complex is formed with apparently the active site lysyl residue of the enzyme. This was established by showing that (a) aldolase activity is destroyed when the enzyme is treated with NaBH₄ in the presence of formaldehyde; (b) the stoichiometry of binding (formaldehyde to enzyme) is 1:1 on a molar basis; (c) formaldehyde competes with the binding of pyruvate or glyoxylate in the presence of NaBH₄; and (d) the isolated reducedenzyme-formaldehyde adduct is N⁵-methyllysine.

Advantage was taken of this finding to show that the irreversible inactivation of 2-keto-4-hydroxyglutarate aldolase by cyanide in the presence of formaldehyde (or, by inference, with any one of a number of other aldehydes) is due to the formation of an aminonitrile. This conclusion is based upon the demonstration that the mole per mole stoichiometry of the complex formed with the enzyme is 1:1:1 (aldolaseformaldehyde-cyanide) and that N⁶-carboxymethyllysine can be isolated after strong acid hydrolysis of the enzyme-[¹⁴C]-formaldehyde-cyanide adduct.

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