Kinetic models for synthesis by a thermophilic alcohol dehydrogenase

Abstract

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 primary alcohols. The enzyme utilizes NADP and NADPH as cosubstrates better than NAD and NADH. The maximum velocities (V(m)) for secondary alcohols at 65 degrees C are 10 to 100 times higher than those at 25 degrees 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-propanol inhibited the oxidation of alcohol competitively with respect to cyclopentanol, and uncompetitively with respect to NADP. Dimethylsulfoxide inhibited the reduction of cyclopentanone competitively with respect to cyclopentanone, and uncompetitively with respect to NADPH. As a product inhibitor, NADP was competitive with respect to NADPH. These results demonstrate that the enzyme binds the nucleotide and then the alcohol or ketone to form a ternary complex which is converted to a product ternary complex that releases product and nucleotide in that order.At 25 degrees C, all aldehydes and ketones examined inhibited the enzyme at concentrations above their Michaelis constants. The substrate inhibition by cyclopentanone was incomplete, and it was uncompetitive with respect to NADPH. Furthermore, cyclopentanone as a product inhibitor showed intercept-linear, slope-parabolic inhibition with respect to cyclopentanol. These results indicate that cyclopentanone binds to the enzyme-NADP complex at high concentrations. The resulting ternary complex slowly dissociates NADP and cyclopentanone.At 65 degrees C, all of the secondary alcohols, with the exception of cyclohexanol, show substrate activation at high concentration. Experiments in which NADP was the variable substrate and cyclopentanol as the constant-variable substrate over a wide range of concentrations gave double reciprocal plots in which the intercepts showed substrate activation and the slopes showed substrate inhibition. These results indicate that the secondary alcohols bind to the enzyme-NADPH complex at high concentrations and that the resulting ternary complex dissociates NADPH faster than the enzyme-NADPH complex.

Biocatalytic process optimization based on mechanistic modeling of cholic acid oxidation with cofactor regeneration

“…The K m for acetone and isopropanol are in the same range reported for the homolog ADH-TB (Ford et al, 1993). Differences can be caused by using progress curves for analysis in the presence of cholic acid.…”

“…For the homologous ADH-TB higher K m values for different substrates are reported, so an extended range up to 10 mM was provided (Ford et al, 1993).…”

“…In our laboratory, we expressed a NADPH-dependent ADH from Thermoanaerobacter ethanolicus (ADH-TE). The amino acids sequence of ADH-TE shares 97% identity to ADH-TB from Thermoanaerobacter brockii which was described to have an ordered bi-bi kinetic mechanism (Ford et al, 1993).…”

Structure‐function relationships in immobilized chymotrypsin catalysis

“…49 Under these conditions, eq. (19) indicates that k cat becomes approximately k cat ≈ k 3 (21) Assuming that this equation also applies for the immobilized enzyme. Table I shows that k cat (ca.…”

“…The reaction rate v obs of immobilized ␣-CT with ATEE was determined in a differential recirculation reactor of the type described by Ford et al 21 equipped with a 10-cm water jacket for temperature control. In a typical assay, 5-10 mg of immobilized enzyme was retained in a Millipore 13-mm ultrafiltration cell by a stainless-steel screen and a cellulose ester filter with mean pore diameters of 8 m. The substrate solution (1 mM ATEE in 0.1 M phosphate buffer, pH 7.3, 1 M NaCl) was passed through the packed-bed reactor into an open reservoir and recirculated by a Cole-Parmer Masterflex variable speed pump through a 1-cm constant temperature flow-through cuvette where the change in substrate absorbance was recorded at 238 nm with a Bausch & Lomb Spectronic 21 UV-Visible spectrophotometer.…”