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However, a SCD mutation, N102S, changed binding of this substrate completely, rendering its prenylation at position 3 impossible (Figure 6D and 6E).
Notably, there is a significant increase in the KM of G3P with the mutant proteins in comparison to the wild type enzyme indicating impaired binding of this substrate.
This V246M mutant mimic also displays a significantly higher K m for the acyl-CoA thioester substrate, suggesting that binding of this substrate is compromised at least subtly in this mutant.
74 Binding of this substrate resulted in a tight complex, which rapidly released the internal water.
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The saturating dependence on substrate concentration and dependence on affinity is reminiscent of the Michaelis Menten model for enzyme kinetics, in which reversible binding of the substrate (in this case, actin monomers) is followed by irreversible conversion to product (F-actin subunits).
In addition, a relatively hydrophobic region can be found parallel to the isoalloxazine ring of FMN; this indicates favorable binding of the substrate's aromatic/hydrophobic part in an orientation that results in a C=C double bond reduction.
Since the peptide substrate Abz-TESE↓SRGAIY-Dpa-KK has eight residues on the prime side, sufficiently long to potentially interact with the Dis domain, we hypothesize that 2D3 is able to interfere with the binding of this peptide substrate.
Our data substantiate the essential role of this highly conserved arginine residue for binding of the substrate in ACADs.
This conformation may facilitate the exofacial binding of the substrate and its translocation to the cytoplasm.
If CT166 targets Rac1, this targeting most likely includes first binding of the substrate Rac1 (and other possible substrate proteins from the Rho subfamily) to CT166.
These data have shown the great diversity of sizes, shapes, and modes of binding of the substrate binding sites of these mammalian cytochromes P450.
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