Publications

• Preparative synthesis of beta-L-malyl-coenzyme A assisted by malyl-coenzyme A synthetase from Pseudomonas AM1.

  Willibald B., Boves H., Holler E.

  beta-L-Malyl-CoA was synthesized from L-malate, CoA, and ATP in the presence of catalytic amounts of L-malyl-CoA synthetase (thiokinase) from Pseudomona AM1, which had been 50-fold purified by protamine sulfate precipitation, ammonium sulfate precipitation, chromatography on DEAE-cellulose, and af ... >> More

  beta-L-Malyl-CoA was synthesized from L-malate, CoA, and ATP in the presence of catalytic amounts of L-malyl-CoA synthetase (thiokinase) from Pseudomona AM1, which had been 50-fold purified by protamine sulfate precipitation, ammonium sulfate precipitation, chromatography on DEAE-cellulose, and affinity chromatography on High Trap Blue in less than 2 days. The homogeneous enzyme was free of L-malyl-CoA lyase and showed 63% homology with succinyl-CoA synthetase from Thermus aquaticus in its N-terminal sequences. Yields of beta-L-[14C]malyl-CoA(1-10 mumol) were 70% before and 65% after purification in 0.1-0.5 mumol portions by high-performance liquid chromatography on a MN Nucleosil 100-7 C8 column. For most biochemical work, the product was partially purified with an overall 45% yield by chromatography on DEAE-Sephacel. The identity of the compound as beta-L-malyl-CoA was verified by chemical and enzymatic tests, and also in comparison with its chemically synthesized counterpart. The enzymatic synthesis, especially of radioactively labeled beta-L-malyl-CoA, is considerably faster, higher in yield, and less problematic than chemical synthesis. << Less

  Anal Biochem 227:363-367(1995) [PubMed] [EuropePMC]

• Malate thiokinase. Evidence for a random site reaction mechanism.

  Surendranathan K.K., Hersch L.B.

  Half-site reactivity in the malate thiokinase reaction was studied by measuring the reaction of enzyme-bound ligands in a series of single turnover experiments. A dimeric (alpha beta)2 enzyme form containing [14C] succinyl-CoA on one alpha beta subunit pair and [3H]succinyl-CoA on the adjacent alp ... >> More

  Half-site reactivity in the malate thiokinase reaction was studied by measuring the reaction of enzyme-bound ligands in a series of single turnover experiments. A dimeric (alpha beta)2 enzyme form containing [14C] succinyl-CoA on one alpha beta subunit pair and [3H]succinyl-CoA on the adjacent alpha beta subunit pair was prepared. Reaction of this enzyme species with ATP or inorganic phosphate resulted in the release of half of the bound succinyl-CoA. The succinyl-CoA released comprised a 50-50 mixture of [14C]- and [3H]succinyl-CoA. Likewise, enzyme containing [32P]phosphate on one alpha beta subunit pair and nonradioactive phosphate on the adjacent alpha beta subunit pair reacted with ADP releasing half of the bound phosphate as a 50-50 mixture of radioactive and nonradioactive phosphate. These results serve to exclude an alternating site mechanism for the malate thiokinase reaction and support a random reaction of liganded subunits. In addition, it has been shown that enzyme containing 1 phosphate/(alpha beta)2 dimer is inactive toward phosphate transfer. However, succinyl-CoA served to activate this enzyme species for phosphate transfer. These results can be explained in terms of subunit asymmetry. The simplest model is one in which subunit asymmetry is induced upon ligand binding. << Less

  J Biol Chem 258:3794-3798(1983) [PubMed] [EuropePMC]