Berti Lab: Enzymatic Transition States and Enzyme Inhibitors

Berti Lab

Antibiotic Targets

Research

Overview
DNA Repair Enzymes
Antibiotic Targets: AroA & MurA
KIEs by NMR
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Docking a Transition State in the Enzyme's Active Site

The TS of pertussis toxin-catalyzed ADP-ribosylation of Gialpha1 is shown. Pertussis toxin exerts its toxic effect by inhibiting protein Gialpha1, by ADP-ribosylating it, using NAD⁺ as the ADP-ribose donor. The TS was determined, and its structure docked into the active site of the crystal structure of pertussis toxin. In the ADP-ribosylation reaction, protein Gialpha1 will bind on top of pertussis toxin, sandwiching NAD⁺ inside. (Scheuring, J., Berti, P. J. & Schramm, V. L. (1998) Biochemistry 37:2748-2758).

The NAD⁺ transition state docked with pertussis toxin. The surface charge is mapped onto the molecular surface of the TS and the enzyme, going from positive to negative.

(1) The cationic oxocarbenium ion in the TS (blue) is directly adjacent to a Glu residue (red) on the enzyme that is conserved in all known bacterial toxin sequences. The carboxylate directly adjacent to the substrate stabilizes the positive charge that is formed at the TS.

(2) The nicotinamide ring goes from positively charged in NAD⁺ to neutral (green) in the TS. The neutral nicotinamide is bound tightly in a hydrophobic binding pocket (salmon) Side chains have been rotated to make nicotinamide visible. It would be energetically unfavourable to bind the positive charged nicotinamide of the substrate in a hydrophobic pocket.

(3) The negatively charged pyrophosphate (red) is juxtaposed with positively charged (blue) Arg and His residues on the protein. This is not specifically a TS-stabilizing interaction because it also occurs in the substrate-enzyme complex.