KINETIC-ANALYSIS OF CARDIOLIPIN SYNTHASE - A MEMBRANE ENZYME WITH 2 GLYCEROPHOSPHOLIPID SUBSTRATES

Mitochondrial cardiolipin synthase catalyzes the transfer of a phospha tidyl moiety from phosphatidyl-CMP (PtdCMP) to phosphatidylglycerol (P tdGro) in the presence of specific divalent cations. The synthase was solubilized from Saccharomyces cerevisiae mitochondria and purified ab out 300-foId. The partially purified enzyme was part of a medium-size, mixed micelle which had to bind to a foreign substrate/detergent mice lle before catalysis could occur. The kinetics of cardiolipin synthase were studied by changing the molar fraction of substrate in the micel les. The enzyme obeyed Michaelis-Menten kinetics in relation to PtdCMP with a K-m of 0.03 mol%. PtdGro caused sigmoidal kinetics with a low apparent affinity. It is speculated that it was involved in docking th e enzyme to the substrate/detergent micelle. Cardiolipin synthase did not catalyze isotope exchange between [C-14]CMP and PtdCMP, virtually excluding a ping-pong catalytic mechanism. Mg2+ stimulated the activit y by increasing the turnover number rather than the substrate affinity , a mechanism which was also found for the Co2+-activation of rat live r cardiolipin synthase. It is concluded that a direct association of t he metal ion and the enzyme forms the active cardiolipin synthase whic h has a very high affinity for PtdCMP and a lower affinity for PtdGro.