1. Substrate analogue CoA derivatives were applied as inhibitors of citrate synthase. Substitution of the acyl-CoA oxygen next to sulfur by hydrogen was without marked influence on the affinity.2. Carboxymethyl-CoA, a structural analogue of enolic acetyl-CoA, was characterized as a transition state analogue by an affinity 100-fold higher than that of acetyl-CoA. K, of the binary inhibitor-enzyme complex was high (230 pM) but that of the ternary inhibitor-oxaloacetate-enzyme complex was 0.07 pM. Both enzyme subunits bound the inhibitor independently, also in the presence of oxaloacetate.3. (3R,S)-3,4-Dicarboxy-3-hydroxybutyl-CoA, an analogue of citryl-CoA, inhibited the overall reaction noncompetitively against acetyl-CoA and against oxaloacetate ; it was a competitive inhibitor against the hydrolysis and cleavage reactions of (3S)-citryl-CoA. Kinetic data suggest that this inhibitor represents an intermediate analogue.4. The results given above indicate conformational changes of the synthase during the catalytic cycle. In the proposed mechanism the free enzyme represents a hydrolase which in the presence of oxaloacetate, by a wellknown conformational change, is converted into a ligase. If both substrates are present, the ligase is reconverted into the hydrolase upon formation of the intermediate, ( [15,16]. These results indicate the presence of one condensation site per subunit but yield no further information. 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. The results presented here Dedicated to Professor Helmut Holzer on occasion of his 60th anniSome of these results are taken from the Thesis of E. Bayer, Tcch-
-EJB 8301721 . Chemically and stereochemically pure (3 S)-citryl-CoA was prepared enzymically and used as a substrate for citrate synthase to investigate the previously determined unexpectedly low rate of hydrolysis of the (3 RS)-substrate. The unnatural R-diastereomer of this mixture is not inhibitory.2. At low enzyme concentrations the rate of citryl-CoA hydrolysis was linear until the reaction went near to completion; the hydrolysis approached Michaelis-Menten kinetics at high enzyme concentrations. In between these concentration extremes a biphasic rate dependence was detectable, where a fast initial phase lasting a few seconds was followed by a slow steady-state phase.3. Citrate synthase was characterized as a hysteretic enzyme existing in two interconvertible forms, which were designated according to their functions as hydrolase E and ligase E'.4. The hysteretic behaviour originates in the cleavage of citryl-CoA to acetyl-CoA and oxaloacetate. This reaction occurs on the ligase form E', which represents a trap for enzyme form E, the hydrolase.5. The conclusions given above are strengthened by the ordinary hydrolysis kinetics of (2S)-malyl-CoA, a substrate that is not subject to cleavage of the C-C bond on the synthase.6. The results satisfy the kinetic criterion for citryl-CoA being an intermediate of the physiological synthase reaction and, therefore, establish the oscillation of the synthase between hydrolase and ligase states during the catalytic cycle.7. A disorganization of these oscillations can be achieved by limited tryptic proteolysis of the synthase.Recent sequence [ 11 and X-ray-crystallographic studies [2] have unravelled the tertiary structure of citrate synthase, made up of two identical subunits with 437 amino acid residues each. 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. The results are presented in this paper. MATERIALS AND METHODS MaterialsCoenzyme A (Ultra Pure) was from P-L Biochemicals GmbH (St Goar). Acetyl-CoA 141, carboxymethyl-CoA EnzymesCitrate synthase, malate dehydrogenase and phosphotransacetylase were from Boehringer (Mannheim). Enzymes delivered in ammonium sulfate suspension were dialyzed (1 5 h) against 0.1 M Tris buffer, pH 8.0, prior to use. Assays of Citrate Synthase ActivitiesThe physiological synthase reaction (assay I), the hydrolysis of citryl-CoA or malyl-CoA (assay 2) and the cleavage of citryl-CoA (assay 3) were determined in the presence of 53'-dithiobis(2-nitrobe...
In order to initiate studies on the structural and functional relationships of the myosin heavy chain, we constructed a full-length complementary DNA encoding the isoform that is found in the fast white muscle of the embryonic chicken. The complementary DNA contained 108 basepairs of its 3'-untranslated region and was preceded by a leader sequence derived from the alfalfa mosaic virus. Similarly, a complementary DNA encoding 963 amino acids which encompass the subfragment-1 of myosin and part of the subfragment-2 was also constructed. Each was inserted into the expression vector pMT2 and transiently transfected into COS-1 cells. Both constructs directed the expression of the respective proteins, each of which was immunogenic. The full-length and subfragment-1 proteins interacted with actin and demonstrated high levels of a K(+)-activated, EDTA-resistant ATPase activity, which is characteristic of myosin.
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