Evidence for co-operativity 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-co-operative 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-249-->Tyr) mutant was studied using the high fluorescence sensitivity of its tryptophan residues Trp-95 and Trp-117 to the binding of coenzyme moieties. Fluorescence quenching studies performed at 25 degrees C show that SsADH exhibits linearity in the NAD(H) binding [the Hill coefficient (h) approximately 1) at pH 9.8 and at moderate ionic strength, in addition to positive co-operativity (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 co-operative below 20 degrees C (h approximately 3) and negatively co-operative at 40-50 degrees C (h approximately 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 degrees C, but exhibits positive and negative co-operativity for NADH oxidation below (h=3.3 at 20 degrees C) and above (h=0.7 at 70-80 degrees C) this range of temperatures respectively. However, N249Y SsADH displays non-co-operative 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 co-operativity. This is the first report of co-operativity in a tetrameric ADH and of temperature-induced co-operativity in a thermophilic enzyme.
Giordano
Antonietta
A
Istituto di Biochimica delle Proteine, Consiglio Nazionale delle Ricerche, Via Marconi 10, I-80125 Napoli, Italy.
Febbraio
Ferdinando
F
Russo
Consiglia
C
Rossi
Mosè
M
Raia
Carlo A
CA
D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*; D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D003067:Coenzymes / Q000737:chemistry* / Q000378:metabolism*; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D021122:Protein Subunits; D012965:Sodium Chloride; D013050:Spectrometry, Fluorescence; D048229:Sulfolobus solfataricus / Q000201:enzymology*
D003067:Coenzymes; D021122:Protein Subunits; D012965:Sodium Chloride; D000426:Alcohol Dehydrogenase