Transcarboxylase: Strucuture, Flexibility and Mechanism

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Transcarboxylase (TC) is a large multi-enzyme complex that catalyzes the transfer of CO2 from methyl malonyl co-enzyme A to pyruvate to form oxaloacetate and propionyl co-enzyme A. The broad objectives of the proposal are to provide structural information for TC in solution and detailed information on the two classes of carboxylation sites found in separate subunits. Since these two carboxyl transfer half reactions and chemically similar to those for biomedically important (de)carboxylase enzymes, such as propionyl-CoA carboxylase and pyruvate carboxylase, knowledge of mechanism for the transcarboxylase active sites will be of value in structure-function analyses of carboxylases implicated in disease states. Transcarboxylase consists of three different kinds of subunit (12S, 5S and 1.3S) and thirty polypeptide chains with an overall molecular weight of 1.2 million Daltons. Since the subunits have been cloned, and can be reassembled, and since stable substrate-subunit complexes can be f ormed, TC offers a rare opportunity to study the structure, assembly and function of a large oligomeric enzyme complex. To exploit this important opportunity the proposal sets three main goals: 1. To probe the chemistry and mechanism of the 12S active site. Raman difference spectroscopy, principally with single crystals of 12S, will be used to define changes in substrate and active site groups upon substrate binding. 2. To probe the chemistry and mechanism of the 5S and 6S active sites. Raman difference spectroscopy will be used to probe the details of substrate-5S interactions, with studies involving single crystals of 5S playing a major role. 3. To map inter-subunit and active site-active site distances in holo TC using fluorescence resonance energy transfer.