¹⁵N‐ and ¹H‐NMR investigations of the active‐site amino acids in semisynthetic RNase S′ and RNase A

Abstract

Extensive "N-NMR investigations of active-site amino acids were made possible by the solid-phase synthesis of the N-terminal pentadecapeptide of RNase A with selectively ' 5N-enriched amino acids. On complexation with S-protein a fully active RNase S' complex was obtained. The "N resonances of the side chains of lysine-7 (NE), glutamine-11 (Ny), and histidine-12 (Nn,z) were studied in the free synthetic peptide, in the RNase S' complex and in the nucleotide complexes RNase S' with 2'CMP, 3'CMP, and S'AMP.The analysis of the 15N-'H couplings, the 15N line broadenings due to proton exchange, and the chemical shift values showed that, while the imidazole ring is directly involved in the peptide-protein interaction, the side chains of Lys-7 and Gln-11 do not contribute to this interaction. In the nucleotide complexes the resonances of His-12 and Gln-11 are shifted downfield. In the 2'CMP complex a doublet for the Nz signal of His-12 indicates a stable H bond between this nitrogen and the phosphate group of the nucleotide. The other nucleotide influence the resonances of the imidazole group much less, possibly due to a slightly different orientation of the phosphate group. The downfield shift of the Gln-11 resonance indicates an interaction between the carbonyl oxygen of the amide group and the phosphate moiety of the nucleotide. The only observable effect of nucleotide complexation on the Lys-7 signal is line broadening due to reduced proton exchange.For comparison with the 15N-NMR titration curves of His-12 in RNase S' the 'H-NMR titration curves of RNase A were also recorded. Both shape and pK values were very similar for the "N and the IH titration curves. An extensive analysis of the protonation equilibria with several fitting models showed that a mutual interaction of the imidazole groups of the active-site histidines results in flat titration curves. The Hill plots of all resonances of the imidazole rings, including the "N resonances, show a small inflection in the pH range 5.8 -6.4. Since the existence of a diimidazole system is most likely in this pH range, the inflection could be interpreted as a disturbance of the mutual electrostatic interaction of the active-site histidines by a partial H-bond formation between the imidazole groups.

Atomic resolution structures of ribonuclease A at six pH values

“…An overall pK a shift of À1.5 pK units, corresponding to a destabilization of the protonated Lys41 of about 8.4 kJ mol À1 , was calculated. This compares well with the measured pK a of 8.9 in solution (Knoblauch et al, 1988). For comparison, pK a shifts were calculated also for the other lysines in the structure and no signi®cant pK a shift was found, apart from Lys7 (pK a shift À1.2 units), which is the closest to Lys41 (Fig.…”

“…The deprotonation of Lys41 requires its pK a to be peculiarly low. An unusually low value of pK a of 8.9 had previously been found for Lys41 in solution (Knoblauch et al, 1988). As shown by the electrostatic potential at the protein surface, calculated at the two extremes of pH*, the active site is left nearly uncharged at basic pH*, whereas a positively charged area generated by Lys7, Arg10 and Arg39 is still present (Figs.…”

Unambiguous Determination of the Ionization State of a Glycoside Hydrolase Active Site Lysine by 1H−15N Heteronuclear Correlation Spectroscopy

“…The amine of Lys302 forms a buried ion pair, and that of Lys47 is hydrogen bonded to the cellobioside. Both lysines are positively charged, as unambiguously demonstrated by the splitting of their 15 N ζ signals into quartets (| J NH | ∼ 75 Hz) in a 1 H− 15 N HSQC spectrum recorded without 1 H decoupling during 15 N evolution. Qualitative insights into the dynamic properties of these lysines are also provided by the deviations of their quartet intensity ratios from that of ∼3:1:1:3 expected for a highly mobile amine.…”

“…In contrast, the peaks from the internal K47 and K302 diminished in intensity due to the slower global tumbling of the enzyme. Furthermore, since this exchange is both specific and general base catalyzed, , reduction of the sample pH to 5.6 led to the detection of at least 10 amines with increased signal intensities (Figure E,F). Each of these yielded a 15 N quartet in a 1 H-coupled HSQC spectrum (with intensity ratios of ∼2:1:1:2, not shown) and must arise from a fully protonated mobile surface lysine or the N-terminal amine.…”

“…In an ongoing effort to dissect the electrostatic basis for the catalytic proficiency of glycoside hydrolases, we are using NMR spectroscopy to determine the ionization states of active site residues in the 34 kDa family 10 catalytic domain, CexCD, from Cellulomonas fimi β-(1,4)-glycosidase Cex (or CfXyn10A) along its double-displacement reaction pathway . When recorded with an atypical upfield 15 N spectral window, the 1 H− 15 N HMQC spectrum of uniformly 15 N-labeled apo-CexCD yielded a single peak at chemical shifts characteristic of an amine ( 1 H ζ 8.1 ppm, 15 N ζ 34.8 ppm; Figure A) . On the basis of a 3D 15 N-edited NOESY experiment, this signal is assigned to K302 .…”