Activation of Sulfolobus solfataricus alcohol dehydrogenase by modification of cysteine residue 38 with iodoacetic acid.
Reaction of thermostable NAD(+)-dependent alcohol dehydrogenase from Sulfolobus solfataricus with iodoacetate at pH 9.0 and 37 degrees C significantly increases the oxidation rate of aliphatic and aromatic alcohols and decreases the reduction rate of aromatic aldehydes. The archaeal ADH is chemically modified and activated in a Michaelis-Menten-type reaction, where one molecule of the reagent binds per active site. NAD+ in micromolar concentration protects the enzyme against the inhibitor in an uncompetitive manner, while imidazole significantly increases the extent of the activation. Carboxymethylation selectively modifies one out of five cysteine residues per subunit, namely, Cys 38, located in the catalytic site, as determined by peptide and sequence analysis, and enhances by up to 25-fold the oxidation rate of benzyl alcohol. Carboxymethylated SsADH is less thermostable and shows a temperature optimum 30 degrees C lower than that of the native enzyme. The carboxymethylated enzyme exhibits a lower affinity toward the oxidized and reduced coenzyme. The dissociation constants for NAD+ and NADH determined at 25 degrees C and pH 8.8 are 60- and 200-fold higher, respectively, compared to the native enzyme. The significant isotope effect in alcohol oxidation suggests that hydride transfer partially limits the turnover rate of the reaction catalyzed by the modified enzyme, whereas the rate-limiting step for the native enzyme is NADH dissociation. Carboxymethylated enzyme probably gives higher maximum velocities of oxidation because of the faster dissociation of the modified enzyme-coenzyme complex.
Raia
C A
CA
Istituto di Biochimica delle Proteine ed Enzimologia, Consiglio Nazionale delle Ricerche, Napoli, Italy.
Caruso
C
C
Marino
M
M
Vespa
N
N
Rossi
M
M
D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*; D000477:Alkylating Agents / Q000494:pharmacology; D002021:Buffers; D003545:Cysteine / Q000737:chemistry; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D011487:Protein Conformation; D016944:Sulfolobus / Q000201:enzymology*; D013696:Temperature
D000477:Alkylating Agents; D002021:Buffers; D007461:Iodoacetates; D009243:NAD; D000426:Alcohol Dehydrogenase; D003545:Cysteine; D019807:Iodoacetic Acid