Lanthanum binding to aqualysin I, a thermostable serine protease, as probed by lanthanum-139 nuclear magnetic resonance spectrometry

Aqualysin I, a heat-stable protease, has two Ca2+-binding sites. The weaker binding site can also bind several di- and tri-valent metal ions including La3+ other than Ca2+. The metal binding to the weaker binding site is esse ntial for the heat stability of the enzyme. A lanthanum-139 NMR study was c onducted to probe the weaker metal-binding site of the enzyme. The spectrum obtained for holo-aqualysin I, which binds 1 mol Ca2+ per mol of the enzym e in the stronger binding site, showed a single Lorentzian line. The linewi dth of the spectrum was unchanged in the titration experiment. These result s indicate that only the aqueous La3+ ion produces an NMR signal, that no L a-139 signal for La3+ bound to the enzyme is evident, and that the exchange of La3+ between the free-ion and protein-bound states is slow on the NMR t ime scale. A binding constant of 2 x 10(4) M-1 was obtained from the titrat ion experiment. (C)1999 Elsevier Science Inc. All rights reserved.