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

Abstract

We have investigated the lysine side chain amines in the 34 kDa catalytic domain from Cellulomonas fimi beta-(1,4)-glycosidase Cex (or CfXyn10A) using 1H-detected 15N heteronuclear correlation NMR spectroscopy. Signals from the 1Hzeta ( approximately 8 ppm) and 15Nzeta ( approximately 35 ppm) of Lys302 in the unmodified enzyme and Lys47 in a trapped cellobiosyl-enzyme intermediate were detected in a 1H-15N HMQC spectrum (pH 6.5 and 30 degrees C). 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 15Nzeta signals into quartets (|1JNH| approximately 75 Hz) in a 1H-15N HSQC spectrum recorded without 1H decoupling during 15N evolution. Qualitative insights into the dynamic properties of these lysines are also provided by the deviations of their quartet intensity ratios from that of approximately 3:1:1:3 expected for a highly mobile amine. On the basis of the observed ratios of approximately 1:1:1:1 for the quartet of Lys302 and approximately 0.5:1:1:0.5 for Lys47, the amine of the latter active site residue is most rigidly positioned. Signals from at least 8 and 10 additional positively charged, mobile amines in Cex were observed at 10 degrees C and pH 6.5 and 5.6, respectively. By using conditions of reduced temperature, slightly acidic pH, and low general base concentrations, as well as water flipback pulses to minimize the effects of hydrogen exchange, 1H-15N correlation experiments provide a sensitive route to directly investigate the charge states and dynamic properties of the N-terminal and side chain amines in proteins and protein complexes.

Catalytic roles of βLys87 in tryptophan synthase: 15N solid state NMR studies

“…[48] Upon liberation of the lysine residue, the chemical shift of the ε-amino nitrogen shows a much less dramatic, but nonetheless informative, dependence on protonation state, varying from 33 to 24 ppm when switched from the protonated to neutral forms, respectively. [49, 50] Recently, we reported the 15 N chemical shift measurement for the linking imine nitrogen in the internal aldimine state of tryptophan synthase, the first such measurement for the internal aldimine state of a PLP-dependent enzyme. [39] Here we extend this initial work and employ 15 N SSNMR combined with enzyme containing 15 N enriched ε-amino Lys residues to directly probe the protonation states of βLys87 for two additional stable intermediates in the β-subunit catalytic cycle.…”

Unambiguous Determination of Protein Arginine Ionization States in Solution by NMR Spectroscopy

“…Although conformational and chemical exchange broadening precludes direct 1 H η detection, a count of the number of protons can nevertheless be made if exchange with the solvent is slow enough to observe 15 N– 1 H spin–spin couplings (Figure S5 in the Supporting Information). This was previously used to unambiguously determine the protonation state for lysine side chains in proteins . In the same vein, Figure c shows a 2D arginine 15 N η/ϵ – 13 C ζ correlation spectrum measured in the absence of 1 H‐decoupling during the 15 N chemical shift encoding period.…”

γ-Hydroxy-1,N2-propano-2′-deoxyguanosine DNA Adduct Conjugates the N-Terminal Amine of the KWKK Peptide via a Carbinolamine Linkage

“…These are formed by the N-terminal amine of lysine; 14 the lysine side-chain amines are protonated at pH 7. 36,52 The existence of imine 9 is surmised because the conjugate can be reduced with NaCNBH 3 . 14 However, these conjugates potentially exist as an equilibrium mixture of carbinolamine 8 , imine 9 , and pyrimidopurinone 10 (Scheme 1).…”