The mechanism of rate-limiting motions in enzyme function

Abstract

The ability to use conformational flexibility is a hallmark of enzyme function. Here we show that protein motions and catalytic activity in a RNase are coupled and display identical solvent isotope effects. Solution NMR relaxation experiments identify a cluster of residues, some distant from the active site, that are integral to this motion. These studies implicate a single residue, histidine-48, as the key modulator in coupling protein motion with enzyme function. Mutation of H48 to alanine results in loss of protein motion in the isotope-sensitive region of the enzyme. In addition, k(cat) decreases for this mutant and the kinetic solvent isotope effect on k(cat), which was 2.0 in WT, is near unity in H48A. Despite being located 18 A from the enzyme active site, H48 is essential in coordinating the motions involved in the rate-limiting enzymatic step. These studies have identified, of approximately 160 potential exchangeable protons, a single site that is integral in the rate-limiting step in RNase A enzyme function.

Fig. 1.

Conformational motions in WT RNase A. (A) ¹⁵N and ¹³C^ε rcCPMG relaxation dispersion data at 14.1 T for 5% (black), 33% (orange), 52% (blue), and 100% (green) ²H₂O. Fitted lines to data points are from single field fits. (B) The dependence of k_ex on the fraction of ²H₂O is shown. Blue, indicates residues for which k_ex does not depend on the atom fraction of ²H₂O, whereas residues that show a decrease in k_ex are shown in black for S16 (circles), S22 (squares), F46 (diamonds), M29 (triangles), M30 (inverted triangles), A109 (open circles), A64 (open squares), and A20 (open diamonds). (C) Proton inventory data showing the dependence of k_ex on the ²H₂O atom fraction from a global analysis of the isotope effect data.

Fig. 2.

Location of flexible residues. Residues involved in chemical exchange are shown as spheres with the amino acid residue number indicated. Gold spheres indicate residues in which a normal ²H solvent isotope effect of 2 is observed. Spheres colored green are atoms that are flexible and do not exhibit a solvent isotope effect. The four histidine residues are depicted as red sticks. Select secondary structure elements are indicated.

Fig. 3.

Solvent isotope effect on enzyme kinetics. Measured k_cat in H₂O (black) and ²H₂O (green) for WT (circles) and H48A (triangles) RNase A.

Fig. 4.

Solvent isotope and mutation effects. ¹⁵N and ¹³C^ε rcCPMG relaxation for 5% (black), 10% (aqua), 52% (blue), and 100% (green) ²H₂O. WT data are indicated by circles, and triangles are used to indicate H48A data. NMR CPMG dispersion data were collected at 18.8 T (open symbols) and 14.1 T (closed symbols) at 298 K.

Fig. 5.

Mutational effects on RNase A. (A) 3′-CMP titration for K31, H12, and A19 in WT and H48A for [CMP]/[RNase A] ratios of 0.0 (red) 0.2 (orange), 0.4 (yellow), 0.7 (green), 1.3 (blue), 2.7 (purple), 6.0 (magenta), and 12 (cyan). Arrows indicate the direction of resonance shift with increasing [CMP]. Residues in A are indicated on the RNase A structure in B. (B) Overlay of apo (gray) and substrate-bound (cyan) RNase A. The substrate analog, phosphothymidine pyrophosphoryl adenosine phosphate (16), is shown as a dotted surface representation. Residues V43 and T45 located on loop β-1 are shown in stick representation along with the corresponding surface contact made with substrate. Hydrogen bonds between H48 and loops 1 and β-1 are shown as black dashed lines. Crystallographically identified water molecules in the vicinity of H48 are shown as small spheres. (C and D) Chemical shift differences, Δ for 3′-CMP binding to WT (C) and H48A (D) RNase A (Δ is defined in SI Materials and Methods).

Fig. 6.

Experimental and simulated CPMG dispersion curves. Curves are shown for F46 (A) and T100 (B) at 14.1 T. The solid black curves represent the WT, experimental curves, with exchange parameters Δω, k_ex, p_A, and R₂⁰ equal to 510 s⁻¹, 1,657 s⁻¹, 0.95, and 15.1 s⁻¹, respectively, for F46 and 356 s⁻¹, 1,657 s⁻¹, 0.95, and 14.4 s⁻¹, respectively, for T100. The solid red, blue, and green curves were simulated by using Eq. 1 and the experimentally determined WT exchange parameters, except p_A = 0.97, 0.99, and 0.999, respectively. The dashed red curve was generated by using Eq. 1 and the experimentally determined WT exchange parameters, with k_ex = 130 s⁻¹.