The long-term objective of this project is to develop a total view of how succinyl-CoA synthetase (SCS) is assembled and how it functions catalytically. In the present application, two lines of approach are proposed. First, site-directed mutagenesis will be performed with the alpha2 beta2 SCS from E. coli, in order to change amino acid residues that are known or strongly suggested to be involved at the active site. The initial residues to be examined are a reactive sulfhydryl group on the Beta-subunit and histidine-246 of the alpha-subunit, site of compulsory phosphorylation during the enzymatic reaction. Three tryptophan residues on the beta-subunit will also be subjected to mutation, in order to explain inactivation of SCS by chemical modification. Plasmid GS131 which contains the genes for the alpha- and Beta- subunits of E. coli SCS will be mutagenized in vitro using oligonucleotides that contain single or double base mismatches. The E. coli deletion strain TK3D18 which does not contain SCS will be transformed with the mutant plasmids. The enzyme protein will be isolated using adaptations of the established purification procedure for the wild type protein. Kinetic analyses will be performed, in order to characterize the type of effect, if any, the mutation has had on the enzyme. It is expected that mutation of alphahis-246 to asn will lower kcat significantly. Chemical derivatives of the alpha-subunit will be prepared and isolated. They will be used to test the alternating sites cooperativity model that has been proposed for this enzyme. The second approach will involve study of the refolding of E. coli SCS and SCS from pig heart, which is an alpha beta protein. The dependency of the refolding of E. coli SCS on ATP will be studied by molecular exclusion in an HPLC system and the use of fluorescent probes. Pig heart SCS which has only recently been observed to refold to give active enzyme for the first time will be examined by circular dichroism spectroscopy in guanidine hydrochloride solutions, as well as by the use of HPLC and fluorescent probes. It will be of interest to determine why the pig heart enzyme does not require nucleoside triphosphate substrate for activation upon refolding.
