Distinctive interactions in the holoenzyme formation for two isoforms of glutamate decarboxylase.

The interactions between glutamate decarboxylase (GAD) and its cofactor pyridoxal phosphate (PLP) play a key role in the regulation of GAD activity. The enzyme has two isoforms, GAD65 and GAD67. A comparison of binding constants, rate constants, and kinetic profiles for the formation of holoenzyme (holoGAD65 and holoGAD67) revealed that the two isoforms interact distinctively with the cofactor. GAD67 exhibits a higher binding constant for PLP binding, making it more difficult to dissociate PLP from holoGAD67 than holoGAD65. Meanwhile, PLP binding occurs at a much slower rate for GAD67 than GAD65, as evidenced by lower rate constants and a slower initial rate of the holoenzyme formation. Job's plots revealed a stoichiometry of 1:1 for PLP binding to GAD65 before and after the saturation level of PLP, while 1:2 for PLP binding to GAD67 prior to the saturation of PLP and 1:1 at the saturation level of PLP. These results suggested that the two binding sites of GAD65 exhibit similar affinities for PLP. In contrast, one binding site of GAD67 exhibits a significantly higher affinity for PLP than the other binding site. Based on these findings, it was proposed that a slower PLP binding to GAD67 than GAD65 and a less ease to dissociate PLP from holoGAD67 than holoGAD65 are important underlying factors. This attributes to GAD67 being more highly saturated by PLP and GAD65 being less saturated by PLP. A larger conformation change constant for GAD67 than GAD65 supported a significant conformational change induced by the initial PLP binding to GAD67, which affects the other binding site affinity of GAD67. The present studies provided valuable insights into distinctive properties between the two isoforms of GAD.

From two-state to three-state: the effect of the P61A mutation on the dynamics and stability of the factor for inversion stimulation results in an altered equilibrium denaturation mechanism.

Factor for inversion stimulation (FIS) is a 22 kDa homodimeric protein found in enteric bacteria that is involved in the stimulation of certain DNA recombination events and transcription regulation of many genes. FIS has a central helix with a 20 degrees kink, which is only reduced by 4 degrees after a proline 61 to alanine mutation (P61A). This mutation appears to have little effect on FIS function, yet it is striking that proline 61 is highly conserved among fis genes. Therefore, we studied the role of proline 61 on the stability and flexibility of FIS. The urea-induced equilibrium denaturation of P61A FIS was monitored by circular dichroism and fluorescence anisotropy. Despite the apparent two-state transition, the concentration dependence of the transition slope (m value) shows that a two-state model, as seen for wild-type (WT) FIS, did not adequately describe the denaturation of P61A FIS. Global fitting of the data indicates that the denaturation of P61A FIS occurs via a three-state process involving a dimeric intermediate and has an overall DeltaG(H2O) for unfolding of 18.6 kcal/mol, 4 kcal/mol higher than that for WT FIS. Limited trypsin proteolysis experiments show that the DNA binding C-terminus of P61A FIS is more labile to cleavage than that of WT FIS, suggesting an increased flexibility of this region in P61A FIS. In contrast, the resulting dimeric core (residues 6-71) of P61A FIS is more resistant to proteolysis, consistent with the presence of a dimeric intermediate not seen in WT FIS. Model transition curves generated using the parameters obtained by global fitting predicted a two-state-like transition at low P61A concentrations that becomes less cooperative with increasing protein concentration, as was experimentally observed. At concentrations of P61A FIS much higher than are experimentally feasible, a biphasic transition is predicted. Thus, this work demonstrates that a single mutation may be sufficient to alter a protein's denaturation mechanism and underscores the importance of analyzing the denaturation mechanism of oligomeric proteins over a wide concentration range. These results suggest that proline 61 in FIS may be conserved in order to optimize the global stability and the dynamics of the functionally important C-terminus.

Equilibrium denaturation studies of the Escherichia coli factor for inversion stimulation: implications for in vivo function.

The Factor for Inversion Stimulation (FIS) is a dimeric DNA binding protein found in enteric bacteria that is involved in various cellular processes, including stimulation of certain specialized DNA recombination events and transcription regulation of a large number of genes. The intracellular FIS concentration, when cells are grown in rich media, varies dramatically during the early logarithmic growth phase. Its broad range of concentrations could potentially affect the nature of its quaternary structure, which in turn, could affect its ability to function in vivo. Thus, we examined the stability of FIS homodimers under a wide range of concentrations relevant to in vivo expression levels. Its urea-induced equilibrium denaturation was monitored by far- and near-UV circular dichroism (CD), tyrosine fluorescence, and tyrosine fluorescence anisotropy. The denaturation transitions obtained were concentration-dependent and showed similar midpoints (C(m)) and m values, suggesting a two-state denaturation process involving the native dimer and unfolded monomers (N(2) <--> 2U). The DeltaG(H(2)O) for the unfolding of FIS determined from global and individual curve fitting was 14.2 kcal/mole. At concentrations <9 microM, the FIS dimer began to dissociate, as noted by the change in CD signal and size-exclusion high-pressure liquid chromatography retention times and peak width. The estimated dimer dissociation constant based on the CD and size-exclusion chromatography data is in the micromolar range, resulting in a DeltaG(H(2)O) of at least 5 kcal/mole less than that calculated from the urea denaturation data. This discrepancy suggests a deviation from a two-state denaturation model, perhaps due to a marginally stable monomeric intermediate. These observations have implications for the stability and function of FIS in vivo.