Significance of sulphhydryl groups in the activity of isocitrate lyase of castor endosperm

Abstract

Titration of isocitrate lyase of germinating castor endosperm, a tetrameric protein (mol. wt. 140 000; subunit mol. wt. 35 000), with 5,5′-dithio-bis-(2-nitrobenzoate) (DTNB) reveals the presence of 4.15 'accessible' sulphhydryl (SH) groups. Denatured enzyme is found to titrate for 7.9 SH groups per molecule. Half of the 'accessible' groups react more readily with DTNB than the remaining half. The enzyme is inactivated at low concentrations of p-chloromercury benzoate (p-CMB) or N-ethylmaleimide (NEM). Under the conditions [p-CMB] ≫ [enzyme], the inactivation reaction is found to be biphasic in which half of the activity is lost more rapidly than the remaining half. With NEM (reagent in excess over enzyme), the activity is destroyed in a single exponential decay (pseudo-first order kinetics). The loss of activity is found to be linearly related to the blocking of accessible SH groups. Glyoxylate protects the enzyme strongly against attack by NEM. Protection by succinate is very much weaker. Significance of these results is discussed. © 1991.