Fig. 4: The activity of HisFH is correlated with the population of the active conformation.

a Methyl TROSY spectra of NMR active HisH (hC84S background) that is incorporated in the HisFH complex either in the presence of saturating concentrations of Gln (black) or in the presence of saturating amounts of Gln and ProFAR (red). Weak signals of the inactive conformation are still present in the HisFH/Gln/ProFAR complex (left), indicating that the complex does not fully adopt the active conformation. The populations of inactive and active conformations are shifted by different HisF ligands and mutations (right, closeups of the hV8 and hV111 signals are shown). The populations of the active conformation are given at the bottom of the spectrum and the signals of the inactive (I) and active (A) conformations are labeled in the spectrum of the WT HisFH complex in the presence of ProFAR. b Methyl TROSY spectra of NMR active HisH in a fully active HisFH complex during catalytic turnover. Close-ups for hV8/hV111 in the presence of Gln/ProFAR during catalytic turnover (red) are shown for WT HisFH (top) and for the hyperactive HisFH–fD98E complex (bottom). The signals of the inactive (I) and active (A) conformations are labeled in the spectra. Under multiple turnover conditions, the active conformation is observed only for the fD98E mutant. Spectra of the inactive conformation (before addition of ProFAR) are shown in black. c Correlation between ln(k_cat) of HisFH and ΔΔG^mut of the active conformation for different mutants/ ligands. A linear fit with a slope of (−RT)⁻¹ is shown (red line). This implies that the glutamine turnover rates in HisFH are directly determined by the population of the active conformation. Data are presented as mean values ± 1 standard deviation (based on 8 resonances (x-axis) or ≥ 28 timepoints(y-axis)). Source data are provided as a Source data file.