Purification and characterization of Helicobacter pylori alcohol dehydrogenase.
Alcohol dehydrogenase of Helicobacter pylori (HPADH) was purified from the soluble fraction of cultured bacteria (strain NCTC 11637) by anion exchange and affinity chromatography. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the 160-fold purified enzyme displayed one protein band with a mobility that corresponded to an M(r) of 38,000. Although HPADH was capable of utilizing both NADP and NAD as cofactors in alcohol oxidation, it showed a strong preference for NADP over NAD. Kinetic studies revealed a Km value of 26 mM and a kcat value of 530 min-1 for ethanol/active site at 37 degrees C in 0.1 M potassium phosphate buffer (pH 7.4). The enzyme was considerably more active toward primary aliphatic alcohols than secondary alcohols. The Km and kcat values decreased as the chain length of the alcohol increased. Benzyl alcohol was a 100 times better substrate than ethanol in terms of kcat/Km values. At neutral pH, HPADH was more effective in aldehyde reduction than in alcohol oxidation. Because of its high specific activity for ethanol (14 units mg-1) under physiological conditions, HPADH can also effectively produce acetaldehyde at higher ethanol levels. This reversed function of HPADH and the production of toxic and reactive acetaldehyde could account for at least some of the gastrointestinal morbidity associated with H. pylori infection.
Research Unit of Alcohol Diseases, University of Helsinki, Finland.
P
P
Kaihovaara
K S
KS
Salmela
R P
RP
Roine
T U
TU
Kosunen
M
M
Salaspuro
D000079:Acetaldehyde / Q000378:metabolism; D000426:Alcohol Dehydrogenase / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D000431:Ethanol / Q000493:pharmacokinetics; D016480:Helicobacter pylori / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010084:Oxidation-Reduction
D000431:Ethanol; D000426:Alcohol Dehydrogenase; D000079:Acetaldehyde