Direct Detection of the Calcium–Sodium–Calcium Metal Triad in Thermostable α-Amylases from Bacillus sp. Bacteria in Aqueous Solution by 23Na Quadrupole-Central-Transition NMR Spectroscopy

Abstract We report that the Na+ ion within the calcium–sodium–calcium (Ca–Na–Ca) metal triad in two thermostable bacterial α-amylases (ca. 58 kDa) can be directly detected by 23Na (I = 3/2) quadrupole-central-transition (QCT) NMR spectroscopy in aqueous solution. We show that 23Na QCT NMR signals from hyperthermostable Bacillus licheniformis α-amylase (BLA) and its mesophilic homologue, Bacillus amyloliquefaciens α-amylase (BAA), exhibit remarkable subppm spectral resolution, which is unprecedented in NMR studies of quadrupolar nuclei in proteins. Using this new 23Na QCT NMR methodology, we examine the pH-dependent unfolding, melting temperatures (Tm), long-term thermal inactivation profiles, and chemical denaturing of BAA and BLA. We also use 23Na QCT NMR to monitor EDTA titration experiments, which yielded the apparent binding constants of the Na+ ion to BAA and BLA to be (0.3 ± 0.1) × 109 and (0.6 ± 0.1) × 109 M–1, respectively. Because the Ca–Na–Ca metal triad is located in the vicinity of the active cleft, the subppm spectral resolution of 23Na QCT NMR makes it possible to detect subtle changes of the enzyme structure caused by the presence of inhibitors such as acarbose and quercetin. This work represents the first time that Na+ ions tightly bound to an enzyme are directly observed by 23Na QCT NMR in aqueous solution. This new 23Na QCT NMR approach can potentially be extended to detection of other metal ions with quadrupolar nuclei such as K+, Mg2+, and Zn2+ in metalloenzymes in their native aqueous environment.