Kinetic analysis of cardiolipin synthase: a membrane enzyme with two glycerophospholipid substrates.
Mitochondrial cardiolipin synthase catalyzes the transfer of a phosphatidyl moiety from phosphatidyl-CMP (PtdCMP) to phosphatidylglycerol (PtdGro) in the presence of specific divalent cations. The synthase was solubilized from Saccharomyces cerevisiae mitochondria and purified about 300-fold. The partially enzyme was part of a medium-size, mixed micelle which had to bind to a foreign substrate/detergent micelle before catalysis could occur. The kinetics of cardiolipin synthase were studied by changing the molar fraction of substrate in the micelles. The enzyme obeyed Michaelis-Menten kinetics in relation to PtdCMP with a Km of 0.03 mol%. PtdGro caused sigmoidal kinetics with a low apparent affinity. It is speculated that it was involved in docking the enzyme to the substrate/detergent micelle. Cardiolipin synthase did not catalyze isotope exchange between [14C]CMP and PtdCMP, virtually excluding a ping-pong catalytic mechanism. Mg2+ stimulated the activity by increasing the turnover number rather than the substrate affinity, a mechanism which was also found for the Co(2+)-activation of rat liver cardiolipin synthase. It is concluded that a direct association of the metal ion and the enzyme forms the active cardiolipin synthase which has a very high affinity for PtdCMP and a lower affinity for PtdGro.
Schlame
M
M
Department of Biological Sciences, Wayne State University, Detroit, Michigan 48202, USA.
Zhao
M
M
Rua
D
D
Haldar
D
D
Greenberg
M L
ML
D000818:Animals; D002384:Catalysis; D002462:Cell Membrane / Q000201:enzymology*; D003035:Cobalt / Q000494:pharmacology; D003567:Cytidine Diphosphate Diglycerides / Q000378:metabolism; D003568:Cytidine Monophosphate / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins; D008823:Micelles; D008928:Mitochondria / Q000201:enzymology; D008930:Mitochondria, Liver / Q000201:enzymology; D010715:Phosphatidylglycerols / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013379:Substrate Specificity; D017855:Transferases (Other Substituted Phosphate Groups) / Q000378:metabolism*
D003567:Cytidine Diphosphate Diglycerides; D008565:Membrane Proteins; D008823:Micelles; D010715:Phosphatidylglycerols; D003035:Cobalt; D017855:Transferases (Other Substituted Phosphate Groups); C100091:cardiolipin synthetase; D003568:Cytidine Monophosphate; D008274:Magnesium