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 anio n exchange and affinity chromatography. On sodium dodecyl sulfate-poly acrylamide gel electrophoresis, the 160-fold purified enzyme displayed one protein band with a mobility that corresponded to an M(r) of 38,0 00. Although HPADH was capable of utilizing both NADP and NAD as cofac tors in alcohol oxidation, it showed a strong preference for NADP over NAD. Kinetic studies revealed a K-m value of 26 mM and a k(cat) value of 530 min(-1) for ethanol/active site at 37 degrees C in 0.1 m potas sium phosphate buffer (pH 7.4). The enzyme was considerably more activ e toward primary aliphatic alcohols then secondary alcohols. The K-m a nd k(cat) values decreased as the chain length of the alcohol increase d. Benzyl alcohol was a 100 times better substrate than ethanol in ter ms of k(cat)\K-m values. At neutral pH, HPADH was more effective in al dehyde reduction than in alcohol oxidation. Because of its high specif ic activity for ethanol (14 units mg(-1)) under physiological conditio ns, HPADH can also effectively produce acetaldehyde at higher ethanol levels. This reversed function of HPADH and the production of toxic an d reactive acetaldehyde could account for at least some of the gastroi ntestinal morbidity associated with H. pylori infection.