Thiol groups of Escherichia coli citrate synthase and their influence on activity and regulation

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The modification of Escherichia coli citrate synthase (citrate oxaloacetate-lyase( pro-3S- CH 2·COO - a ̊ acetyl-CoA, EC 4.1.3.7 ) with 5,5′-dithiobis-(2-nitrobenzoic acid) has been investigated. 1. (1)|In low ionic strength (20 mM Tris - HCl, pH 8.0): (a) Eight thiol groups per tetramer of the native enzyme reacted with Nbs 2. (b) Two of the eight accessible thiols were modified rapidly with the loss of 26% enzyme activity but with no change in the NADH inhibition. The remaining six were modified more slowly, resulting in a further 60% loss of activity and complete desensitization to NADH. (c) The 2nd-order rate constant for the modification of the rapidly reacting thiols in 2.5·10 4 M -1. At the reagent concentrations used (0.1 to 0.2 mM) the modification of the six thiols in the slow kinetic set appeared to be 1st-order; at 0.1 mM dithionitrobenzoic acid their rate of modification was approximately 30 times slower than the thiols in the fast kinetic set. 2. (2)|In high ionic strength (20 mM Tris·HCl, pH 8.0, 0.1 M KCl): (a) Four thiol groups were modified in a single kinetic set and it appeared that these thiols are four of the six slowly modified in the absence of KCl. (b) The modification resulted in 70% loss of enzyme activity and complete loss of NADH inhibition. 3. (3)|From the kinetic analysis it is proposed that the four thiol groups accessible to dithionitrobenzoic acid in the absence and presence of 0.1 M KCl are those involved in the response to NADH. Modification of any one of these four groups produced no reduction in the inhibition; instead, loss of NADH sensitivity was coincident with the appearance of tetrameric protein possesing three substituted thiols, whereas enzyme with one or two modified groups was still fully inhibited by NADH.