Homology Modelling of BaeJ KS

In an effort to rationalise the substrate selectivity exhibited by BaeJ KSl and examine the effect of an Asn residue at the X position, a homology model of KS 1 was constructed using the same methodology as in Chap. 3. Due to the shorter nature of the predicted intermediate, it was possible to model the native acyl chain of 27 into the binding pocket. Using a previously published structure of a FA FAS complex (PDB 1EK4) [9] as a guide, the acyl chain of 27 was successfully modelled into the active site of KS 1. 

A further finding from the homology modelling was the putative existence of a pocket in the KS binding cleft which was able to accommodate the terminus of 

To test the influence of the Asn residue upon KSl acylation rate, a BaeJ KS1(N206A) mutant was constructed. Upon initial incubation of KS1(N206A) with SNAC 27, markedly reduced levels of acylation were observed compared to WT KSl, with the initial rate reduced by 65 % (Table 4.1 and Fig. 4.9). Approximately the same reduction in acylation rate was observed for the other nitrogen-containing SNACs 28, 30 and 34, indicating the mutation may well have removed a favourable interaction between the Asn side-chain and the nitrogen of 

In order to gain more insight into the interaction between the active site Asn206 and SNAC 27, a Michaelis-Menten ueatment was performed on WT KSl and KS1(N206A). This analysis required incubation of KS domains with a range of SNAC 27 concentrations (0.250-2 mM), with the measured acylation velocities used to construct Lineweaver-Burk plots (Fig. 4.10). 

The Michaelis-Menten analysis yielded a Km value of 1.8 iiiM 0.4 for WT KSl. In comparison, the Km value for KS1(N206A) was 3.7 iiiM 1.3, suggesting a decreased affinity towards SNAC 27 upon removal of the active site Asn residue. Additionally, the kcat/Km value was reduced by a factor of 2, implying that the catalytic efficiency of KS1(N206A) has also been reduced by removal of the Asn residue. It is noteworthy that this reduction in kcat/Km is largely due to the difference in Km values, as kcat values for WT and mutant are extremely similar (Table 4.2). 

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