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[(super 1)H] and [(super 15)H] correlation spectroscopy is used to examine the lysine amines in CexCD and its trapped glycosyl-enzyme intermediate, demonstrating clearly that K47 interacts with the cellobiosyl moiety via its positively charged side chain. It is shown that in cursory survey, well-resolved amine signals were observed in the [(super 1)H]- [(super 15)H] HMQC spectra of ubiquitin, RanGAP, and a xylanase.

The article investigates the lysine amines in CexCD and the trapped glycosyl-enzyme intermediate with the use of NMR spectroscopy to determine the ionization states of active site residues. It demonstrates that K47 interacts with the cellobiosyl moiety though its positively charged side chain and that CexCD has 18 lysines exposed on the surface of the protein. Amines that remain undetected in NMR was because of the optimization of spectral parameters for amides.

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.