The conformational stabilities of native pig citrate synthase (PCS), a recombinant wild-type PCS, and six active-site mutant pig citrate synthases were studied in thermal denaturation experiments by circular dichroism and in urea denaturation experiments by using DTNB to measure the appearance of latent SH groups. His274 and Asp375 are conserved active-site residues in pig citrate synthase that bind to substrates and are implicated in the catalytic mechanism of the enzyme. By site-directed mutagenesis, His274 was replaced with Gly and Arg, while Asp375 was replaced with Gly, Asn, Glu, or Gln. These modifications were previously shown to result in 10(3)-10(4)-fold reductions in enzyme specific activities. The thermal unfolding of pig citrate synthase and the six mutants in the presence and absence of substrates showed large differences in the thermal stabilities of mutant proteins compared to the wild-type pig citrate synthase. The functions of His274 and Asp375 in ligand binding were measured by oxalacetate protection against urea denaturation. These data indicate that active-site mutations that decrease the specific activity of pig citrate synthase also cause an increase in the conformational stability of the protein. These results suggest that specific electrostatic interactions in the active site of citrate synthase are important in the catalytic mechanism in the chemical transformations as well as the conformational flexibility of the protein, both of which are important for the overall catalytic efficiency of the enzyme. Study holds ProTherm entries: 332, 333, 334, 335, 336, 337, 338 Extra Details:
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:15 p.m.
|Number of data points||31|
|Proteins||Citrate synthase, mitochondrial ; Citrate synthase, mitochondrial|
|Assays/Quantities/Protocols||Experimental Assay: activity units:% ; Experimental Assay: activity units:units/mg ; Experimental Assay: Tm ; Experimental Assay: activity_km ; Experimental Assay: activity_kd ; Derived Quantity: dTm|
|Libraries||Mutations for sequence ASSTNLKDILADLIPKEQARIKTFRQQHGNTVVGQITVDMMYGGMRGMKGLVYETSVLDPDEGIRFRGYSIPECQKMLPKAKGGEEPLPEGLFWLLVTGQIPTEEQVSWLSKEWAKRAALPSHVVTMLDNFPTNLHPMSQLSAAITALNSESNFARAYAEGIHRTKYWELIYEDCMDLIAKLPCVAAKIYRNLYREGSSIGAIDSKLDWSHNFTNMLGYTDAQFTELMRLYLTIHSDHEGGNVSAHTSHLVGSALSDPYLSFAAAMNGLAGPLHGLANQEVLVWLTQLQKEVGKDVSDEKLRDYIWNTLNSGRVVPGYGHAVLRKTDPRYTCQREFALKHLPHDPMFKLVAQLYKIVPNVLLEQGKAKNPWPNVDAHSGVLLQYYGMTEMNYYTVLFGVSRALGVLAQLIWSRALGFPLERPKSMSTDGLIKLVDSK|