KINETIC-MODELS FOR SYNTHESIS BY A THERMOPHILIC ALCOHOL-DEHYDROGENASE

Alcohol dehydrogenase (E.C. 1.1.1.1) from Thermoanaerobium brockii at 25-degrees-C and at 65-degrees-C is more active with secondary than pr imary alcohols. The enzyme utilizes NADP and NADPH as cosubstrates bet ter than NAD and NADH. The maximum velocities (V(m)) for secondary alc ohols at 65-degrees-C are 10 to 100 times higher than those at 25-degr ees-C, whereas the K(m) values are more comparable. At both 25-degrees -C and 65-degrees-C the substrate analogue 1,1,1,3,3,3-hexafluoro-2-pr opanol inhibited the oxidation of alcohol competitively with respect t o cyclopentanol, and uncompetitively with respect to NADP. Dimethylsul foxide inhibited the reduction of cyclopentanone competitively with re spect to cyclopentanone, and uncompetitively with respect to NADPH. As a product inhibitor, NADP was competitive with respect to NADPH. Thes e results demonstrate that the enzyme binds the nucleotide and then th e alcohol or ketone to form a ternary complex which is converted to a product ternary complex that releases product and nucleotide in that o rder. At 25-degrees-C, all aldehydes and ketones examined inhibited th e enzyme at concentrations above their Michaelis constants. The substr ate inhibition by cyclopentanone was incomplete, and it was uncompetit ive with respect to NADPH. Furthermore, cyclopentanone as a product in hibitor showed intercept-linear, slope-parabolic inhibition with respe ct to cyclopentanol. These results indicate that cyclopentanone binds to the enzyme-NADP complex at high concentrations. The resulting terna ry complex slowly dissociates NADP and cyclopentanone. At 65-degrees-C , all of the secondary alcohols, with the exception of cyclohexanol, s how substrate activation at high concentration. Experiments in which N ADP was the variable substrate and cyclopentanol as the constant-varia ble substrate over a wide range of concentrations gave double reciproc al plots in which the intercepts showed substrate activation and the s lopes showed substrate inhibition. These results indicate that the sec ondary alcohols bind to the enzyme-NADPH complex at high concentration s and that the resulting ternary complex dissociates NADPH faster than the enzyme-NADPH complex.