Investigation of the impact of point mutations on the binding kinetics of ScpA-hC5a pair

Insight into the mode of interaction of an enzyme with its substrate can help us to design and target drugs or inhibitor efficiently. This thesis explores the role of charged residues present at enzyme-substrate (ScpA-hC5a) interface in the binding kinetics. The kinetic and thermodynamic parameters for the ScpA-hC5a complex were measured by surface plasmon resonance (SPR). These studies showed that C5a was bound with high affinity (33.7 nM) to ScpA and that the majority of interactions were provided by hC5acore.

The mutation of positively charged residues; R37, R40 and R46 of hC5a destabilise mutated complexes due to a decrease in the rate of association combined with a more substantial increase in the rate of dissociation. This indicated that these residues might be involved initially in long-range interactions which form the initial encounter complex and then in short range interactions involved in the formation of a high affinity complex.

The effect on binding upon replacement of negatively charged residues; E864, D889 and D783 in the Fn2 domain of ScpA were also investigated. Mutation of E864 and D889 to alanine in ScpA showed similar binding parameters as the wild-type interaction. However, substitution of D783 with alanine reduced the binding affinity significantly impacting significantly on the rate of dissociation. Therefore D783 could be an important residue at the binding interface and predominantly involved in short-range interactions.

Double mutant cycle experimets revealed that E864 of interacted favourably with R37 and R46 while D889 interacted favourably with R37, R40 and R46 of hC5a. D783 has favourable coupling with R40 (KD = 1400 nM) and R46 (KD = 539.3 nM) of hC5a. This indicated the interactions between these distantly located residues might be mediated by solvent or counter residues. Increased ionic strength had significant impact only on the association rate indicating that electrostatic steering significantly contributes to the binding.

The proposed 4-step model showed parental and mutated ScpA(S512A) has decreased preference for mutated hC5a. The slow dissociation of the product (k4) (hC5acore) from ScpA could be the rate-limiting step and could lead to product inhibition. Fast dissociation (k4) was observed for ScpA(S512A)D783A indicating that k4 might not be rate limiting step in this case and faster product clearance could lead to higher catalytic efficiency.

The map of important exosite residues which has significant impact on the enzyme-substrate binding can help us to design more effective drugs which can target the exosite along with the active site.