Biochemical properties of alcohol dehydrogenase from Drosophila lebanonensis

Abstract

Purified Drosophila lebanonensis alcohol dehydrogenase (Adh) revealed one enzymically active zone in starch gel electrophoresis at pH 8.5. This zone was located on the cathode side of the origin. Incubation of D. lebanonensis Adh with NAD+ and acetone altered the electrophoretic pattern to more anodal migrating zones. D. lebanonensis Adh has an Mr of 56,000, a subunit of Mr of 28 000 and is a dimer with two active sites per enzyme molecule. This agrees with a polypeptide chain of 247 residues. Metal analysis by plasma emission spectroscopy indicated that this insect alcohol dehydrogenase is not a metalloenzyme. In studies of the substrate specificity and stereospecificity, D. lebanonensis Adh was more active with secondary than with primary alcohols. Both alkyl groups in the secondary alcohols interacted hydrophobically with the alcohol binding region of the active site. The catalytic centre activity for propan-2-ol was 7.4 s-1 and the maximum velocity of most secondary alcohols was approximately the same and indicative of rate-limiting enzyme-coenzyme dissociation. For primary alcohols the maximum velocity varied and was much lower than for secondary alcohols. The catalytic centre activity for ethanol was 2.4 s-1. With [2H6]ethanol a primary kinetic 2H isotope effect of 2.8 indicated that the interconversion of the ternary complexes was rate-limiting. Pyrazole was an ethanol-competitive inhibitor of the enzyme. The difference spectra of the enzyme-NAD+-pyrazole complex gave an absorption peak at 305 nm with epsilon 305 14.5 X 10(3) M-1 X cm-1. Concentrations and amounts of active enzyme can thus be determined. A kinetic rate assay to determine the concentration of enzyme active sites is also presented. This has been developed from active site concentrations established by titration at 305 nm of the enzyme and pyrazole with NAD+. In contrast with the amino acid composition, which indicated that D. lebanonensis Adh and the D. melanogaster alleloenzymes were not closely related, the enzymological studies showed that their active sites were similar although differing markedly from those of zinc alcohol dehydrogenases.

Drosophila Alcohol Dehydrogenase: Acetate–Enzyme Interactions and Novel Insights into the Effects of Electrostatics on Catalysis

“…In the case of His254, this distance is for the His in subunit B and the Tyr in subunit A. This larger positive net electrostatic potential for DlADH compared to DmADH-S is also reflected in a slightly faster electrophoretic migration towards the cathode at pH 8.5 in starch gel electrophoresis, 46 and in a higher pI value obtained from isoelectric focusing. 46,48 The coenzyme binding site All the residues that directly interact with the coenzyme via a hydrogen bond are 100% conserved in DmADH-S and DlADH.…”

“…This is in agreement with previous kinetic studies on these two enzymes, which showed that they have almost identical substrate specificities, and may also explain the small differences seen in catalytic efficiency of some secondary alcohols, and in their dissociation constants (K EO,I ) of the two alcohol-competitive inhibitors pyrazole and trifluoroethanol. 34,45,46 Another interesting difference between DmADH-S and DlADH is the ionization of a single amino acid residue that regulates the binding of alcohols and alcohol-competitive inhibitors to the active site in the binary enzyme-NAD C complex. The pK a value of this residue was approximately 0.5 pH unit lower in the DlADH than in DmADH-S. 25,27,37 This suggests a slightly more positive electrostatic character of the active site in DlADH than in DmADH-S, as suggested previously.…”

Drosophila lebanonensis ADH: analysis of recombinant wild‐type enzyme and site‐directed mutants

“…Enzymatic determinations: activity inhibition, kinetic parameters, pH profiles and thermal stability ADH activity was measured spectrophotometrically by the increase in absorbance at 340 nm, using propan-2-01 as substrate and NAD' as cofactor in 20 mM Tris-HCl pH 8.6 [16]. For activity inhibition determinations, total protein extract from the bacteria1 cultures was incubated with or without mAb LLBEI [14] for 1 h at 37°C before measuring activity.…”

Well ordered crystals of a short-chain alcohol dehydrogenase from Drosophila lebanonensis: re-evaluation of the crystallographic data and rotation-function analysis

“…Biochemical data suggest that the protein is a dimer with the molecular mass 54 kDa composed of two identical subunits (Winberg et al, 1986) under the conditions of a gel-filtration experiment. The size of the unit cell is compatible with the presence of a subunit dimer in the asymmetric unit of the cell, which corresponds to a packing density of 2.15 A 3 Da-1.…”