Redox-elimination reaction catalyzed by yeast alcohol dehydrogenase

Yeast alcohol dehydrogenase (EC 1.1.1.1) catalyzed reduction of N,N-dimethyl-4-nitrosoaniline by NADH. The stoichiometry of reaction, steady-state kinetic parameters, and the pH-profile for this reaction were estimated. On that basis, the minimal mechanism of the above reaction was postulated.     

The role of hydrogen bonding in the enzymatic reaction catalyzed by HIV-1 protease

The hydrogen-bond network in various stages of the enzymatic reaction catalyzed by HIV-1 protease was studied through quantum-classical molecular dynamics simulations. The approximate valence bond method was applied to the active site atoms participating directly in the rearrangement of chemical bonds. The rest of the protein with explicit solvent was treated with a classical molecular mechanics model. Two possible mechanisms were studied, general-acid/general-base (GA/GB) with Asp 25 protonated at the inner oxygen, and a direct nucleophilic attack by Asp 25. Strong hydrogen bonds leading to spontaneous proton transfers were observed in both reaction paths. A single-well hydrogen bond was formed between the peptide nitrogen and outer oxygen of Asp 125. The proton was diffusely distributed with an average central position and transferred back and forth on a picosecond scale. In both mechanisms, this interaction helped change the peptide-bond hybridization, increased the partial charge on peptidyl carbon, and in the GA/GB mechanism, helped deprotonate the water molecule. The inner oxygens of the aspartic dyad formed a low-barrier, but asymmetric hydrogen bond; the proton was not positioned midway and made a slightly elongated covalent bond, transferring from one to the other aspartate. In the GA/GB mechanism both aspartates may help deprotonate the water molecule. We observed the breakage of the peptide bond and found that the protonation of the peptidyl amine group was essential for the peptide-bond cleavage. In studies of the direct nucleophilic mechanism, the peptide carbon of the substrate and oxygen of Asp 25 approached as close as 2.3 A.     

Stereospecificity of hydrogen transfer in the saccharopine dehydrogenase reaction.

The stereospecificity of hydrogen transfer in the synthesis of saccharopine from alpha-ketoglutarate and L-lysine catalyzed by saccharopine dehydrogenase (N5-(1,3-dicarboxypropyl)-L-lysine: NAD oxidoreductase (L-lysine-forming), EC 1.5.1.7) was examined by using [4A-3H]- and [4B-3H]NADH. The enzyme showed the A-stereospecificity. The NMR analysis of the saccharopine prepared with [4"A-2H]NADH revealed that the label was incorporated into the C-2 of the glutaryl moiety.     

Enzymatic hydrogen transfer reduction of α-chloro aromatic ketones catalyzed by a hyperthermophilic alcohol dehydrogenase

An alcohol dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus (PFADH) effectively catalyzed the reductions of various substituted α-chloroacetophenones to furnish the corresponding (R)-configurated α-chlorohydrins with excellent enantiomeric purity. The co-factor NADH could be recycled with d-glucose dehydrogenase/d-glucose system or in a coupled substrate approach using iso-propanol as the hydrogen donor. The hydrogen transfer mode should be more cost-effective. Thus, the PFADH-catalyzed hydrogen transfer reductions of some substrates were carried out on the preparative scale, demonstrating that this enzyme would be a valuable biocatalyst for the preparation of chiral chlorohydrins of pharmaceutical interest.     

Stereochemistry of the hydrogen transfer to NADP catalyzed by D-galactose dehydrogenase from Pseudomonas fluorescens.

The stereochemistry of the hydrogen transfer to NADP catalyzed by D-galactose dehydrogenase (EC 1.1.1.48) from P. fluorescens was investigated. The label at C-1 of D-[1-³H] galactose was enzymatically transferred to NADP and the resulting [4-³H]NADPH was isolated and its stereo-chemistry at C-4 investigated. It was found that the label was exclusively located at the 4(S) position in NADPH which calls for classification as a B-enzyme. The correlation of this finding with tentative classification rules of NAD(P)-linked dehydrogenases in regard to their stereo-chemistry of hydrogen transfer to the coenzyme is discussed.     

Stereochemistry of the hydrogen transfer to NAD catalyzed by D-galactose dehydrogenase from Pseudomonas fluorescens.

The stereochemistry of the hydrogen transfer to NAD catalyzed by D-galactose dehydrogenase (E.C. 1.1.1.48) from P. fluorescens was investigated. The label at C-1 of D-[1--3H] galactose was enzymatically transferred to NAD and the resulting [4--3H]NADH was isolated and its stereochemistry at C-4 investigated. It was found that the label was exclusively located at the 4(S) position in NADH which calls for classification as a B-enzyme. This result was confirmed by an alternate approach in which [4--3H]NAD was reduced by D-galactose as catalyzed by D-galactose dehydrogenase. The sterochemistry at C-4 of the nicotinamide ring would then have to opposite to that in the first experiment. As expected, the label was now exclusively located in the 4(R) position, again confirming the B-calssification of the enzyme.     

Pressure variation of enzymatic reaction rates: yeast and liver alcohol dehydrogenase.

The kinetics of yeast and liver alcohol dehydrogenase (YADH and LADH) have been investigated by spectrophotometry at pressures between 1 and 2000 bar. For YADH the common random two substrate mechanism has been used as a model for evaluation of the pressure variation of five kinetic constants in the ethanol-NAD reaction. The dissociation volume associated with each constant is estimated and it is found that the dissociation of binary complexes is followed by large volume decreases, while the dissociation of ternary complexes is followed by smaller volume increases. There is a volume increase following formation of the activated complex in the rate determining step, and the over-all reaction rate decreases with pressure, going to zero at 2000 bar. LADH shows a complicated behaviour at high pressure. This is believed to be due to the substrate inhibition phenomenon occurring at ethanol concentrations above 10 mM. At such concentrations the reaction rate increases with pressure, reaching a maximum at about 1200 bar and goes to zero at 2500 bar. At ethanol concentrations lower than 10 mM there is a small decrease of reaction rate with pressure. To relate the volume Changes of the over-all process to those of the intermediate complexes, the partial molal volume of ethanol, acetaldehyde, NAD⁺ and NADH are determined by density measuraments.