The Prominent Role of Serines 302/307 in the Activity and Stability of Human Caspase9: Appraisal of the S302D and S307D Variants.

Abstract

Caspase-9 is crucial for initiating apoptosis, and its activity is tightly regulated through various mechanisms, especially phosphorylation by kinases activated by extracellular growth factors, osmotic stress, or during mitosis. Mass spectrometric analyses have shown that residues S302 and S307 in human caspase-9 are prone to phosphorylation. To investigate the effects of phosphorylation at these sites, three phosphomimetic variants of recombinant caspase-9 were created: S302D, S307D, and the combined S302D/S307D variant. The QuickChange method was employed to generate these mutant constructs, which were expressed in Escherichia coli (E. coli) and purified using affinity chromatography. For enzymatic assays, the chromogenic substrate Ac-LEHD-pNA was utilized, and the temperature profiles of enzyme activity were assessed. Computational modeling was used to predict the structures of the mutants, allowing for comparison with the native enzyme. The results indicated that both the S302D and S302D/S307D variants exhibited complete loss of enzyme activity. In contrast, the S307D variant demonstrated a 10-fold increase in the Michaelis constant (K_m) for the substrate and a 4-fold increase in the maximum reaction rate (V_max) compared to the wild-type enzyme. Notably, the k_cat/K_m value for wild-type caspase-9 was three times greater than that of the S307D variant. The optimal temperature for wild-type activity was between 30 and 37 °C, while for the S307D variant, it ranged from 37 to 45 °C. Importantly, the S302 residue is essential for caspase-9 function; introducing a negative charge at this position leads to complete inactivation of the enzyme.