The possible role of a threonine residue in a highly conserved sequence of phosphoenolpyruvate carboxylase

Phosphoenolpyruvate carboxylase (PEPCase) is an allosteric enzyme involved in regulating the flow of carbon into the tricarboxylic acid (TCA) cycle for some organisms. It has been observed previously that a mutation in the amino acid sequence at position 188 of the wild-type enzyme in which threonine is changed to isoleucine results in a loss of its activation by fructose 1,6-bis phosphate (FBP). Threonine 188 is a highly conserved amino acid residue among a series of PEPCases, but any connection to FBP-mediated activation is brought into question by the fact that the activity of many of them is not regulated by FBP. The objective of the research reported here is to obtain more information about the role this amino acid plays in allosteric regulation of the FBP responsive enzyme. To do this, site-directed mutagenesis has been done resulting in the following substitutions for threonine at position 188: isoleucine (T188I), tryptophan (T188W), cysteine (T188C), lysine (T188K) and serine (T188S). This study focuses on the partial characterization of the serine mutant T188S. Isoelectric focusing showed that the T188S mutant enzyme has the same pI as the wild-type. Similar experiments with the other mutants showed that only the T188K, which is catalytically inactive, has a different pI. Initial kinetic analyses of the T188S mutant shows that it is allosterically activated by both acetyl coenzyme A and FBP, but, unlike the wild-type enzyme, activation by these two regulators is not synergistic. Analyses of the partially purified mutant enzyme in sucrose gradients in the absence of regulators, and in the presence of aspartate, or complete reaction mixture minus added bicarbonate, was done to determine the aggregation state of the enzyme. It appears that the mutant enzyme T188S showed the same sedimentation pattern as the wild-type enzyme. From this experiments it is inferred that the enzyme is dimeric in the absence of the regulators and tetrameric in the presence of 5 mM aspartate. It appears that the regulation of the T188S mutant enzyme is different from the wild-type but that both show similar physicochemical properties.