Chemical modification of citrate synthase

Hysteretic Behaviour of Citrate Synthase. Alternating Sites during the Catalytic Cycle

“…The subunits were shown to consist of a large and a small domain in two different crystal forms, designated as open and closed, and interconvertible by an 18" rotation of the small domain relative to the large one. In agreement with our results [3,4] the sites (ligase and hydrolase) previously considered to be separated [5,61 faded into one active site, supposed to correspond to the closed form; the open form was suggested to function in product release. Before knowledge of the structural work we have proposed a 'closed' form (ligase) to alternate with an 'open' form (hydrolase) during the catalytic cycle such that binding of oxaloacetate to the latter form generates the ligase, and consumption of oxaloacetate with formation of citryl-CoA regenerates the hydrolase [4].…”

“…The subunits were shown to consist of a large and a small domain in two different crystal forms, designated as open and closed, and interconvertible by an 18" rotation of the small domain relative to the large one. In agreement with our results [3,4] Ever since the demonstration that the synthase catalyzes the hydrolysis and cleavage reactions of citryl-CoA [7], its intermediate formation from acetyl-CoA and oxaloacetate was questionable because the rate of hydrolysis of the applied (3 RS)-substrate was lower than that of the physiological overall reaction. Chemically and stereochemically pure (3 S)-citryl-CoA unexpectedly low hydrolysis rate.…”

“…During the burst phase of the citryl-CoA hydrolysis x increases from catalytic cycle to cycle whereas the ES state decreases. Owing to the decreasing concentration the contribution of this state towards the formation of free enzyme E becomes smaller with time until it reaches a constant residual value, formed by the redistribution of free enzyme E according to Eqn (3). At this time nearly all of the enzyme population exists in the E'S state and the isomerization process is complete.…”

Evidence from Inhibitor Studies for Conformational Changes of Citrate Synthase

“…During attempts to use diazoacetyl-CoA as a photoaffinity label for the synthase it was found that the main product of the photolytic reaction, carboxymethyl-CoA (I1 1) [19], was a most powcrful inhibitor. As shown in Fig.…”

“…Thus, the reaction could occur at two separated sites (ligase and hydrolase); the subunits could operate independently or cooperate in a 'flip-flop' mechanism as suggested for dimeric enzymes [17,18]. It appears unlikely that both partial reactions, electrophilic substitution with inversion of configuration, and hydrolysis, could occur at one and the same active site but experimental evidence indicates that only one site exists [19]. We have therefore assumed that this active site during substrate turnover switches between two forms, one representing the ligase, the other one the hydrolase activity.…”