John S. McKinley-McKee

Alcohol dehydrogenase from the fruitfly Drosophila melanogaster Substrate specificity of the alleloenzymes AdhS and AdhUF

The substrate specificity of the two alleloenzymes AdhS an AdhUF from Drosophila melanogaster has been studied and found to be similar. With most of the secondary alcohols, the Vm value is essentially the same, and indicative of a Theorell-Chance mechanism with rate-limiting enzyme-coenzyme dissociation. The experiments indicate that the enzyme-coenzyme complex formed with AdhUF dissociates at a faster rate than the corresponding complex with AdhS. For primary alcohols the Vm value is much lower than for secondary alcohols, varies with the type of alcohol and the dissociation of the enzyme-coenzyme complex is not rate limiting. For these alcohols a primary isotope effect with deuteroethanol indicates that it is the interconversion of the ternary complexes that is rate determining. Studies with the enantiomers of butan-2-ol and octan-2-ol show that both alkyl groups in the secondary alcohols interact hydrophobically with the alcohol-binding region of the active site. However, the two parts of the alcohol-binding region that interact with the two alkyl groups are of different size. The high activity observed with secondary alcohols and especially with (R)-(+)-cis-verbenol, indicates that these flies can metabolize terpenes. Such compounds may be part of the pheromone system in the flies with D. melanogaster alcohol dehydrogenase playing a role in pheromone metabolism.

Alcohol dehydrogenase from the fruitfly Drosophila melanogaster Inhibition studies of the alleloenzymes AdhS and AdhUF

Different metal binding inhibitors of horse liver alcohol dehydrogenase, similarly affect the Drosophila melanogaster AdhS and AdhUF alleloenzymes. However, binding is generally weaker and the experiments show that the alleloenzymes although not zinc metalloenzymes, behave to the metal binding reagents very much as if they were. The metal-directed, affinity-labelling, imidazole derivative BrImPpOH reversibly inhibits, but does not inactivate the alleolenzymes. This confirms there is no active site metal atom with cysteine as a metal ligand, as found in zinc alcohol dehydrogenases. Pyrazole is a strong ethanol-competitive inhibitor of AdhS and AdhUF alleloenzymes. Formation of the ternary enzyme-NAD-pyrazole complex gives an absorption increase between 295-330 nm. This enables an active site titration to be performed and the determination of epsilon (305 nm) of 15.8 . 10(3) M-1 . cm-1. Inhibition experiments with imidazole confirm that with secondary alcohols such as propan-2-ol, a Theorell-Chance mechanism predominates, but with ethanol and primary alcohols, interconversion of the ternary complexes is rate limiting. Salicylate is a coenzyme competitive inhibitor and KEI suggests that the coenzyme adenosine binding region is similar is Drosophila and horse liver alcohol dehydrogenase. Drosophila alcohol dehydrogenase is found not to form a ternary complex with NADH and isobutyramide. In this and other properties it is like carboxymethyl liver alcohol dehydrogenase. Both Drosophila and carboxymethyl alcohol dehydrogenase bind coenzyme in a similar manner to native horse liver alcohol dehydrogenase, but substrate binding differs between each. Inhibition by Cibacrone blue, indicates that amino acid 192 which is lysine in AdhS and threonine in AdhUF, is located in the coenzyme-binding region. Proteolytic activity present in preparations of alcohol dehydrogenase from D. melanogaster, is considered due to a metalloprotease, for which BrImPpOH is a potent inactivator.

The alcohol dehydrogenase alleloenzymes AdhS and AdhF from the fruitfly Drosophila melanogaster: an enzymatic rate assay to determine the active-site concentration.

A rapid and reproducible enzymatic rate assay for the quantitative determination of the concentration of active sites is presented for the alleloenzymes AdhS and AdhF from Drosophila melanogaster. Using this procedure the turnover numbers as catalytic-center activities were found to be 12.2 sec−1 for AdhF and 3.4 sec−1 for AdhS with secondary alcohols. This showed a slower dissociation of the coenzyme from the binary enzyme-NADH complex with AdhS and hence a stronger binding of NADH to this alleloenzyme. With ethanol, the catalytic-center activity was 1.4 sec−1 for AdhS and 2.8 sec−1 for AdhF, and hence the single amino acid mutation distinguishing the two alleloenzymes also affected hydride transfer.

Drosophila melanogaster alcohol dehydrogenase: Substrate sterospecificity of the AdhF alleloenzyme

Abstract The substrate specificity and the stereospecificity of the AdhF alleloenzyme from Drosophila melanogaster have been investigated. These properties like the topology of the alcohol binding region and the mechanism of oxidation of primary and secondary alcohols are found to be similar to that of the AdhS and AdhUF alleloenzymes. AdhF like AdhUF, binds the coenzyme-competitive inhibitor Cibacron blue much more weakly than does the AdhS alleloenzyme. This shows that amino acid residue 192 affects coenzyme binding. It also accounts for the observed differences between the activities of these alleloenzymes with respect to their maximum velocity ( V m ) with secondary alcohols as substrates.

The reactivity of affinity labels: A kinetic study of the reaction of alkyl halides with thiolate anions—a model reaction for protein alkylation

Abstract Factors have been investigated which govern the electrophilic reactivity of alkyl halides with thiolate anions in aqueous solution. In the series of alkyl halides studied, some are potential metal-directed affinity labels, while others are frequently used in protein modification. Previous data on the kinetics of this type of alkylation are compared with the present results. The influence of electronic, polar, and steric factors on alkyl halide reactivity is seen. The following order of reactivity for alkyl halides bearing different α substituents was observed: RCH 2 CH(X)COOCH 3 > RCH 2 CH(X)CONH 2 > RCH 2 CH(X)COOH > RCH 2 CH 2 X > RCH 2 CH(X)CH 2 OH. The metal-directed affinity labels are imidazole derivatives, some of which have substituents in their imidazole ring. The effect of the imidazole ring and of ring substitution on reactivity is seen. The nucleophilic reactivity of thiols is highly pH dependent since the thiolate anion (RS − ) is the reactive species, but only minor differences emerged between different free thiolates.

Kinetic interpretations of active site topologies and residue exchanges in Drosophila alcohol dehydrogenases

1. A comparison of full and partly sequenced Adhs from various Drosophila species reveal that 127 of their 253-255 positions are identical (50% identity). 2. Fifty-six of the 115 C-terminal amino acids building up the alcohol binding region differ. In spite of the large differences in primary structure of the alcohol binding region in the Adh enzyme in distantly related Drosophila species, the substrate specificity and stereospecificity have been retained. The topology of the alcohol binding region has been largely conserved during evolution. 3. The primary structures of the alcohol dehydrogenases (Adh) in the Sophophora subgenus is distinguished by few amino acid exchanges, and kinetic and activity parameters show that those at positions 14, 82, 192 and 214 are directly or indirectly involved in coenzyme binding. 4. In these non-metallo Adhs, a tyrosine has been tentatively identified as a nucleophilic catalyst of the hydride transfer step. The three tyrosines at positions 63, 152 and 178 are conserved among the Drosophila alcohol dehydrogenases.

Substrate specificity of sheep liver sorbitol dehydrogenase

The substrate specificity of sheep liver sorbitol dehydrogenase has been studied by steady-state kinetics over the range pH 7-10. Sorbitol dehydrogenase stereo-selectively catalyses the reversible NAD-linked oxidation of various polyols and other secondary alcohols into their corresponding ketones. The kinetic constants are given for various novel polyol substrates, including L-glucitol, L-mannitol, L-altritol, D-altritol, D-iditol and eight heptitols, as well as for many aliphatic and aromatic alcohols. The maximum velocities (kcat) and the substrate specificity-constants (kcat/Km) are positively correlated with increasing pH. The enzyme-catalysed reactions occur by a compulsory ordered kinetic mechanism with the coenzyme as the first, or leading, substrate. With many substrates, the rate-limiting step for the overall reaction is the enzyme-NADH product dissociation. However, with several substrates there is a transition to a mechanism with partial rate-limitation at the ternary complex level, especially at low pH. The kinetic data enable the elucidation of new empirical rules for the substrate specificity of sorbitol dehydrogenase. The specificity-constants for polyol oxidation vary as a function of substrate configuration with D-xylo> D-ribo > L-xylo > D-lyxo approximately L-arabino > D-arabino > L-lyxo. Catalytic activity with a polyol or an aromatic substrate and various 1-deoxy derivatives thereof varies with -CH2OH > -CH2NH2 > -CH2OCH3 approximately -CH3. The presence of a hydroxyl group at each of the remaining chiral centres of a polyol, apart from the reactive C2, is also nonessential for productive ternary complex formation and catalysis. A predominantly nonpolar enzymic epitope appears to constitute an important structural determinant for the substrate specificity of sorbitol dehydrogenase. The existence of two distinct substrate binding regions in the enzyme active site, along with that of the catalytic zinc, is suggested to account for the lack of stereospecificity at C2 in some polyols.

Drosophila melanogaster alcohol dehydrogenase: An electrophoretic study of the AdhS, AdhF, and AdhUF alleloenzymes

The nature and the interconversion of the three multiple forms Adh-5, Adh-4, and Adh-3 of the purified alleloenzymes AdhS, AdhF, and AdhUF from the fruitflyDrosophila melanogaster have been examined. The experiments show that these multiple forms differ from those in crude extracts of flies homozygous at the Adh locus. On electrophoresis in a starch gel containing NAD or NADH, of purified AdhS which consists of the three Adh forms S-5, S-4, and S-3, five enzymatically active zones appear. This contrasts with the single active zone that arises with crude extracts. Of the five zones that appear with purified enzyme, S-5 gives rise to one, while the other four zones come from the two minor forms S-4 and S-3. The occurrence of the three multiple forms Adh-5, Adh-4, and Adh-3 for each of the purified alleloenzymes is considered due to Adh-5 and, in the case of Adh-4 and Adh-3, deamidation of Adh-5, with the Adh-3 fraction also containing some reversible modified Adh-5. Of the labile amides, at least one must be located in the coenzyme binding region with deamidation preventing coenzyme binding. Pure NAD does not convert Adh-5 to Adh-3 and Adh-1. To produce conversion, the presence of either acetone or butanone along with NAD is necessary. Increased amounts of either acetone or butanone result in increased conversion. In contrast to this, none of the carbonyl compounds cyclohexanone, (+)- and (−)-verbenone, acetaldehyde, acrolein, or crotonaldehyde produces conversion. The ketone group binds to the alcohol binding site in the enzyme-NAD complex. Conversion is considered due to the ketone group binding to a nucleophilic amino acid residue and forming a bridge to the C-4 of the nicotinamide moiety of NAD.

Drosophila lebanonensis alcohol dehydrogenase: pH dependence of the kinetic coefficients.

The alcohol dehydrogenase (ADH) from Drosophila lebanonensis shows 82% positional identity to the alcohol dehydrogenases from Drosophila melanogaster. These insect ADHs belong to the short-chain dehydrogenase/reductase family which lack metal ions in their active site. In this family, it appears that the function of zinc in medium chain dehydrogenases has been replaced by three amino acids, Ser138, Tyr151 and Lys155. The present work on D. lebanonensis ADH has been performed in order to obtain information about reaction mechanism, and possible differences in topology and electrostatic properties in the vicinity of the catalytic residues in ADHs from various species of Drosophila. Thus the pH dependence of various kinetic coefficients has been studied. Both in the oxidation of alcohols and in the reduction of aldehydes, the reaction mechanism of D. lebanonensis ADH in the pH 6-10 region was consistent with a compulsory ordered pathway, with the coenzymes as the outer substrates. Over the entire pH region, the rate limiting step for the oxidation of secondary alcohols such as propan-2-ol was the release of the coenzyme product from the enzyme-NADH complex. In the oxidation of ethanol at least two steps were rate limiting, the hydride transfer step and the dissociation of NADH from the binary enzyme-NADH product complex. In the reduction of acetaldehyde, the rate limiting step was the dissociation of NAD+ from the binary enzyme-NAD+ product complex. The pH dependences of the kon velocity curves for the two coenzymes were the opposite of each other, i.e. kon increased for NAD+ and decreased for NADH with increasing pH. The two curves appeared complex and the kon velocity for the two coenzymes seemed to be regulated by several groups in the free enzyme. The kon velocity for ethanol and the ethanol competitive inhibitor pyrazole increased with pH and was regulated through the ionization of a single group in the binary enzyme-NAD+ complex, with a pKa value of 7.1. The kon velocity for acetaldehyde was pH independent and showed that in the enzyme-NADH complex, the pKa value of the catalytic residue must be above 10. The koff velocity of NAD+ appeared to be partly regulated by the catalytic residue, and protonation resulted in an increased dissociation rate. The koff velocity for NADH and the hydride transfer step was pH independent. In D. lebanonensis ADH, the pKa value of the catalytic residue was 0.5 pH units lower than in the ADHS alleloenzyme from D. melanogaster. Thus it can be concluded that while most of the topology of the active site is mainly conserved in these two distantly related enzymes, the microenvironment and electrostatic properties around the catalytic residues differ.

Methylglyoxal and the polyol pathway: Three-carbon compounds are substrates for sheep liver sorbitol dehydrogenase

Methylglyoxal, 1,2‐propanediol and glycerol are shown to be substrates for sheep liver sorbitol dehydrogenase. With 1,2‐propanediol the enzymecatalyzed reaction occurs specifically with the R(−)‐enantiomer. The maximum velocities and the specificity constants obtained for the three‐carbon substrates are considerably lower than those reported previously for sorbitol, and suggest that rate‐determination is imposed by catalytic steps other than the enzyme‐coenzyme product dissociation. The present findings are discussed in terms of substrate specificity and stereospecificity, and may indicate novel aspects of sorbitol dehydrogenase function in relation to glucose metabolism and diabetic pathogenesis.