Increase of enzyme activity through specific covalent modification with fragments

Abstract

Modulation of enzyme activity is a powerful means of probing cellular function and can be exploited for diverse applications. Here, we explore a method of enzyme activation where covalent tethering of a small molecule to an enzyme can increase catalytic activity (k_cat/K_M) up to 35-fold. Using a bacterial glycoside hydrolase, BtGH84, we demonstrate how small molecule "fragments", identified as activators in free solution, can be covalently tethered to the protein using Michael-addition chemistry. We show how tethering generates a constitutively-activated enzyme-fragment conjugate, which displays both improved catalytic efficiency and increased susceptibility to certain inhibitor classes. Structure guided modifications of the tethered fragment demonstrate how specific interactions between the fragment and the enzyme influence the extent of activation. This work suggests that a similar approach may be used to modulate the activity of enzymes such as to improve catalytic efficiency or increase inhibitor susceptibility.

Fig. 1. Tether design and chemical structures: (a) Structure of BtGH84 (grey) in complex with 3 (yellow) and PUGNAc (green) from PDB:; 4UR9. Model of designed acrylamide linker is shown in orange, demonstrating the possibility of linking fragment 3 to an introduced cysteine at the position of Y550. (b) Chemical structures for the BtGH84 inhibitors PUGNAc (1) and thiamet-G (2), activator 3 and tethering compound 4.

Fig. 2. Activity and inhibitor binding of BtGH84_TM-4 conjugate: (a) structure of the 4MU-GlcNAc substrate used in this study. (b) Michaelis–Menten plot for the hydrolysis of 4MU-GlcNAc by BtGH84_TM-4 (pink), in comparison to native enzyme (green), unmodified BtGH84 (orange) and the non-covalent activator 3 (purple). (c) Representative ITC data for PUGNAc binding to BtGH84_TM (left) and BtGH84_TM-4 (right).

Fig. 3. Covalent activator chemical structures and X-ray crystal structures of BtGH84_TM conjugates. (a) Chemical structures of the six additional acrylamide containing compounds used to label the BtGH84_TM mutant. (b) Stereo image of the BtGH84_TM-4 conjugate (grey) X-ray crystal structure with PUGNAc (green carbons) bound to the active site. The covalent modification is highlighted (pink carbons) and the SA Fo-Fc omit map of PUGNAc and 4 is shown as green mesh contoured at 3.5σ. (c) Stereo image of the BtGH84_TM-8 conjugate (grey) X-ray crystal structure with PUGNAc (green carbons) bound to the active site. The covalent modification is highlighted (blue carbons) and the SA Fo-Fc omit map of PUGNAc and 8 is shown as green mesh contoured at 3.5σ.

Fig. 4. Effect of Y137F mutation and buffer pH on BtGH84 activation. (a) Fold increase in specificity constant over the parent enzyme for BtGH84_TM and BtGH84_QM fragment conjugates. BtGH84_TM and BtGH84_QM activation are shown as green and orange bars respectively. (b) and (c) BtGH84_TM (green), BtGH84_TM-6 (orange), and BtGH84_TM-7 (blue) activity across pH range 4.3–9.0. Activity in (b) is individually normalised to the highest activity for each conjugate for ease of comparison. Activity in (c) is normalised to BtGH84_TM at pH 5.5.