TY - JOUR
T1 - Amine inversion in proteins. A 13C-NMR study of proton exchange and nitrogen inversion rates in...
AU - Goux, W. J.
AU - Teherani, Jane
AU - Sherry, A. Dean
N1 - Funding Information:
A.D.S. gratefully acknowledges support by Grant AM-16947 from the National Institute of Arthritis, Metabolism and Digestive Diseases, National Institute of HeaIth, Education, and Welfare, and Grant AT-584 from the Robert A. Welch Foundation. W.J.G. acknowledges support by Grant AT-885 from the Robert A. Welch Foundation.
PY - 1984/6
Y1 - 1984/6
N2 - Title full: Amine inversion in proteins. A 13C-NMR study of proton exchange and nitrogen inversion rates in Nε{lunate},Nε{lunate},Nα ,Nα-[13C]tetramethyllysine, Nε{lunate},Nε{lunate},Nα ,Nα-[13C]tetramethyllysine methyl ester, and reductively methylated concanavalin A. Exchange rates were calculated as a function of pH from line widths of methylamine resonances in 13C-NMR spectra of Nε{lunate},Nε{lunate},Nα ,Nα-[13C]tetramethyllysine (TML) and Nε{lunate},Nε{lunate},Nα ,Nα-tetramethyllysine methyl ester (TMLME). The pH dependence of the dimethyl α-amine exchange rate could be adequately described by assuming base-catalyzed chemical exchange between two diastereotopic methyl populations related by nitrogen inversion. Deprotonation of the α-amine was assumed to occur by proton transfer to (1) OH-, (2) water, (3) a deprotonated amine or (4) RCO2-. Microscopic rate constants characterizing each of these transfer processes (k1, k2, k3 and k4, respectively) were determined by fitting the rates calculated from line width analysis to a steady-state kinetic model. Using this procedure it was determined that for both TML and TMLME k2=1-10 M-1 s-1, k3=106 M-1 s-1 and ki, the rate constant for nitrogen inversion was about 108-109 s-1. Upper limits of 1012 and 103 M-1 s-1 could be determined for k1 and k4, respectively. A similar kinetic analysis was used to explain pH-dependent line-broadening effects observed for the N-terminal dimethylalanyl resonance in 13C-NMR spectra of concanavalin A, reductively methylated using 90% [13C]formaldehyde. From exchange data below pH 4 it could be determined that amine inversion was limited by the proton transfer rate to the solvent, with a rate constant estimated at 20 M-1 s-1. Above pH 4, exchange was limited by proton transfer to other titrating groups in the protein structure. Based upon their proximity, the carboxylate side chains of Asp-2 and Asp-218 appear to be likely candidates. The apparent first-order microscopic rate constant characterizing proton transfer to these groups was estimated to be about 1 × 104 s-1. Rate constants characterizing nitrogen inversion (ki), proton transfer to OH- (k1) and proton transfer to the solvent (k2) were estimated to be of the same order of magnitude as those determined for the model compounds. On the basis of our results, it is proposed that chemical exchange processes associated with base-catalyzed nitrogen inversion may contribute to 15N or 13C spin-lattice relaxation times in reduclively methylated peptides or proteins.
AB - Title full: Amine inversion in proteins. A 13C-NMR study of proton exchange and nitrogen inversion rates in Nε{lunate},Nε{lunate},Nα ,Nα-[13C]tetramethyllysine, Nε{lunate},Nε{lunate},Nα ,Nα-[13C]tetramethyllysine methyl ester, and reductively methylated concanavalin A. Exchange rates were calculated as a function of pH from line widths of methylamine resonances in 13C-NMR spectra of Nε{lunate},Nε{lunate},Nα ,Nα-[13C]tetramethyllysine (TML) and Nε{lunate},Nε{lunate},Nα ,Nα-tetramethyllysine methyl ester (TMLME). The pH dependence of the dimethyl α-amine exchange rate could be adequately described by assuming base-catalyzed chemical exchange between two diastereotopic methyl populations related by nitrogen inversion. Deprotonation of the α-amine was assumed to occur by proton transfer to (1) OH-, (2) water, (3) a deprotonated amine or (4) RCO2-. Microscopic rate constants characterizing each of these transfer processes (k1, k2, k3 and k4, respectively) were determined by fitting the rates calculated from line width analysis to a steady-state kinetic model. Using this procedure it was determined that for both TML and TMLME k2=1-10 M-1 s-1, k3=106 M-1 s-1 and ki, the rate constant for nitrogen inversion was about 108-109 s-1. Upper limits of 1012 and 103 M-1 s-1 could be determined for k1 and k4, respectively. A similar kinetic analysis was used to explain pH-dependent line-broadening effects observed for the N-terminal dimethylalanyl resonance in 13C-NMR spectra of concanavalin A, reductively methylated using 90% [13C]formaldehyde. From exchange data below pH 4 it could be determined that amine inversion was limited by the proton transfer rate to the solvent, with a rate constant estimated at 20 M-1 s-1. Above pH 4, exchange was limited by proton transfer to other titrating groups in the protein structure. Based upon their proximity, the carboxylate side chains of Asp-2 and Asp-218 appear to be likely candidates. The apparent first-order microscopic rate constant characterizing proton transfer to these groups was estimated to be about 1 × 104 s-1. Rate constants characterizing nitrogen inversion (ki), proton transfer to OH- (k1) and proton transfer to the solvent (k2) were estimated to be of the same order of magnitude as those determined for the model compounds. On the basis of our results, it is proposed that chemical exchange processes associated with base-catalyzed nitrogen inversion may contribute to 15N or 13C spin-lattice relaxation times in reduclively methylated peptides or proteins.
KW - Dimethylaied concanavalin A
KW - NMR exchange kinetics
KW - Nitrogen inversion
KW - Reductively dimeihylated anine
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U2 - 10.1016/0301-4622(84)87019-2
DO - 10.1016/0301-4622(84)87019-2
M3 - Article
C2 - 6430360
AN - SCOPUS:0021441459
SN - 0301-4622
VL - 19
SP - 363
EP - 373
JO - Biophysical Chemistry
JF - Biophysical Chemistry
IS - 4
ER -