Treatment of lactate dehydrogenase (LDH) with o-phthalaldehyde resulted in a time-dependent loss of enzyme activity. The inactivation followed pseudo first-order kinetics over a wide range of the inhibitor. The second-order rate constant for the inactivation of LDH was estimated to be l.52 (mol/L)-1·s-1. The modified enzyme showed a characteristic fluorescence emission spectrum with a maximum at 405 nm upon excitation at 337 nm, consistent with the formation of isoindole derivatives by the cross-linking of proximal cysteine and lysine residues. The loss of enzyme activity was concomitant with the increase in absorbance at 337 nm. Stoichiometric study of the reaction showed that complete loss of activity was accompanied by formation of approximately four moles of isoindole derivatives per mole of LDH subunits. One of the substrates, NADH, partially prevented the enzyme from reacting with o-phthalaldehyde, whereas the other substrate, pyruvate, did not provide any protection. Protection experiments suggest that one of the cysteine-lysine pairs modified by o-phthalaldehyde is near the NADH binding site of LDH.
|Original language||English (US)|
|Number of pages||6|
|Journal||Tsinghua Science and Technology|
|State||Published - Aug 2003|
- Lactate dehydrogenase (LDH)
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