Paracatalytic modification of aldolase: a side reaction of the catalytic cycle resulting in irreversible blocking of two active-site lysyl residues.

Paracatalytic enzyme modifications result from the oxidation of enzyme-substrate carbanions by extrinsic oxidants. During the oxidation of enzyme-activated substrates, transiently reactive intermediates are generated which, without being released from the enzyme, modify groups at the active site. For enzymes producing carbanion intermediates, the combination of the normal substrate with a suitable electron acceptor has thus been proposed as a highly specific binary system for their active site-directed modification. In this study, the structural features of paracatalytically modified fructose-1,6-bisphosphate aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13) from rabbit muscle have been elucidated. This enzyme is completely inactivated within 60 min in the presence of fructose 1,6-bisphosphate in saturating concentration and 0.5 mM hexacyanoferrate(III) (pH 7.6, 25 degrees C). The inactivation is caused by covalent incorporation of one triosephosphate derivative per subunit. Peptide analysis showed that the triosephosphate derivative forms an intrachain crosslink between lysine-146 and lysine-227. According to previous independent experimental evidence, both lysyl residues are located at the active site: the epsilon-amino group of lysine-227 forms a Schiff base intermediate with the carbonyl group of the substrate [Lai, C. Y., Nakai, N. & Chang, D. (1974) Science 183, 1204-1206] and alkylation of lysine-146 by the affinity labeling reagent N-bromoacetylethanolamine phosphate inactivates the enzyme [Hartman, F. C. & Brown, J. P. (1976) J. Biol. Chem. 251, 3057-3062]. The present data thus establish paracatalytic modification as a mode of active site-directed enzyme modification.