Evidence for cooperativity in coenzyme binding to tetrameric Sulfolobus solfataricus alcohol dehydrogenase and its structural basis: fluorescence, kinetic and structural studies of the wild type enzyme and non cooperative N249Y mutant

The interaction of coenzyme with thermostable homotetrameric NAD(H)-dependent alcohol dehydrogenase from the thermoacidophilic sulphur-dependent crenarchaeon Sulfolobus solfataricus (SsADH) and its N249Y (Asn->249Tyr) mutant was studied using the high fluorescence sensitivity of its tryptophans Trp-95 and Trp-117 to the binding of coenzyme moieties. Fluorescence quenching studies performed at 25 oC show that SsADH exhibits linearity in the NAD(H) binding (the Hill coefficient h close to 1) at pH 9.8 and at moderate ionic strength, in addition to positive cooperativity (h = 2.0-2.4) at pH 7.8 and 6.8, and at pH 9.8 in the presence of salt. Furthermore, NADH binding is positively cooperative below 20 oC (h close to 3) and negatively cooperative at 40-50 oC (h close to 0.7), as determined at moderate ionic strength and pH 9.8. Steady-state kinetic measurements show that SsADH displays standard Michaelis-Menten kinetics between 35 and 45 oC, but exhibits positive and negative cooperativity for NADH oxidation below (h = 3.3 at 20 oC) and above (h = 0.7 at 70-80 oC) this range of temperatures, respectively. However, N249Y SsADH displays non-cooperative behaviour in coenzyme binding under the same experimental conditions used for the wild type enzyme. In loop 270-275 of the coenzyme domain and segments at the interface of dimer A/B, analyses of the wild type and mutant SsADH structures identified the structural elements involved in the intersubunit communication and suggested a possible structural basis for cooperativity. This is the first report of cooperativity in a tetrameric alcohol dehydrogenase and of temperature-induced cooperativity in a thermophilic enzyme.