A dynamic knockout reveals that conformational fluctuations influence the chemical step of enzyme catalysis - PubMed (original) (raw)

A dynamic knockout reveals that conformational fluctuations influence the chemical step of enzyme catalysis

Gira Bhabha et al. Science. 2011.

Abstract

Conformational dynamics play a key role in enzyme catalysis. Although protein motions have clear implications for ligand flux, a role for dynamics in the chemical step of enzyme catalysis has not been clearly established. We generated a mutant of Escherichia coli dihydrofolate reductase that abrogates millisecond-time-scale fluctuations in the enzyme active site without perturbing its structural and electrostatic preorganization. This dynamic knockout severely impairs hydride transfer. Thus, we have found a link between conformational fluctuations on the millisecond time scale and the chemical step of an enzymatic reaction, with broad implications for our understanding of enzyme mechanisms and for design of novel protein catalysts.

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Figures

Fig. 1

Fig. 1

Conformational changes that occur during the E. coli DHFR catalytic cycle. (A) Left, cartoon representation of E:NADP+:FOL crystal structure (1RX2, model for the Michaelis complex, E:NADPH:DHF) in the closed conformation. Right, crystal structure of E:NADP+:ddTHF (1RX4, model for the product complex) in the occluded conformation. NADP+ is shown in orange; FOL is shown in yellow and ddTHF in magenta. Red, Met20 loop in the closed conformation; blue, Met20 loop in the occluded conformation. The sites of mutation, N23 and S148 are shown as spheres. (B) Intermediates in the wild type E. coli DHFR catalytic cycle. Intermediates shown in red are in the closed conformation, and those in blue are in the occluded conformation. Prior to hydride transfer the Met20 loop is in the closed conformation, where it packs tightly against the nicotinamide ring of NADP+. Following hydride transfer, the Met20 loop adopts the occluded conformation, in which the nicotinamide ring of NADP+ is sterically hindered from binding in the active site. NADP+ undergoes a concurrent conformational change in which the nicotinamide ring is expelled from the binding pocket, initiating NADP+ release from the ternary product complex. The rate of hydride transfer in the wild type and N23PP/S148A mutant enzyme is indicated in black and green, respectively. Note that the mutation alters the pathway utilized for product and NADP+ release, as shown in Fig. S2.

Fig. 2

Fig. 2

The three-dimensional structures of the E:NADP+:FOL complexes of the N23PP/S148A mutant and wild-type E. coli DHFR are almost identical. (A) Superposition of the crystal structures of wild-type E. coli DHFR (1RX2) and N23PP/S148A E. coli DHFR. Wild-type E. coli DHFR is shown in red, with yellow ligands and N23PP/S148A E. coli DHFR is shown in purple, with green ligands. (B) Active site configuration for wild-type ecDHFR (1RX2-re-refined). Folate is colored yellow, NADP+ orange, the waters are shown as green spheres, and key active-site residues are in blue sticks. (C) Active site configuration for N23PP/S148A ecDHFR. Colors are the same as for B. For clarity, only the major conformation of the glutamate moiety of folate is shown. The active site configurations are almost identical for wild type and N23PP/S148A ecDHFR, including placement of polar residues, key hydrophobic residues and waters, showing that the electrostatic nature of the active site is unchanged by the mutations.

Fig. 3

Fig. 3

The Met20 loop of N23PP/S148A E. coli DHFR remains in the closed position across the chemical step. (A) 1H-15N HSQC spectra of wild-type E:NADP+:FOL (model Michaelis complex, black) in the closed conformation and E:NADP+:THF (product complex, red) in the occluded conformation. Large chemical shift differences are observed, particularly for residues in regions that undergo the closed-to-occluded structural change. Chemical shift changes from closed (black) to occluded (red) are indicated by arrows for several residues in the active site loops. (B) 1H-15N HSQC spectra of N23PP/S148A E:NADP+:FOL (model Michaelis complex, black) and E:NADP+:THF (product complex, red). The 1H-15N HSQC spectra for the complexes of the mutant enzyme are similar; the cross peaks corresponding to the active site loops do not shift, and appear in the position corresponding to the closed conformation for both complexes. A quantitative chemical shift analysis is presented in Fig. S8.

Fig. 4

Fig. 4

Millisecond timescale dynamics in the active site loops of the E:NADP+:FOL complex are impaired by the N23PP/S148A mutation. (A) Representative 15N R2 relaxation dispersion curves for wild type (black), N23PP/S148A (red), N23PP (blue) and S148A (magenta) E:NADP+:FOL. Dispersion data were collected at pH 7.6 and at 301 K. Top, active site loop residues (11, 95, 121), for which the dispersion observed in wild type E:NADP+:FOL is not observed in either N23PP/S148A or N23PP E:NADP+:FOL. S148A retains dispersion for residue 11 and other active site residues, shown in Fig. S11. Bottom, C-terminal associated residues (129, 131, 133), for which dispersion is observed for the wild type and mutant proteins. Residues which display 15N relaxation dispersion in the E:NADP+:FOL complex are mapped onto the structure for wild type (B), N23PP/S148A (C) and S148A (D). 1RX2 coordinates are used for representations in B and D. Red spheres indicate active site-associated residues for which R2 dispersion is observed. Blue spheres indicate C-terminal-associated residues for which R2 dispersion is observed. NADP+ is shown as orange sticks, FOL as yellow sticks. For clarity, only the major conformation of the glutamate moiety of folate is shown.

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