• Archives of Pharmacal Research
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• v.20 no.2
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• pp.115-121
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• 1997

Ile-269 in horse liver alcohol dehydrogenase isoenzyme S(HLADH-S) was mutated to serine by phosphorothioate-based site-directed mutagenesis in order to study the role of the residue in coenzyme binding. The specific activity of the mutant(1269S) enzyme to ethanol was increased 49-fold. All turnover numbers of 1269S enzyme toward 9 primary alcohols were increased. The mutant enzyme showed 3.6, 4.6, 11.6-fold higher catalytic efficiency for $5{\beta}$-androstane-3, 17-dione, $5{\beta}$-cholanic acid-3-one and retinal than wild-type, respectively. The reaction mechanism of 1269S enzyme was ordered bi bi as wild-type's. These results indicate that the hydrophobic interaction of Ile-269 residue with coenzyme plays an important role in dissociation of coenzyme from enzyme-coenzyme complex, which has been known as the rate limiting step of ADH reaction.

• Archives of Pharmacal Research
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• v.15 no.3
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• pp.229-233
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• 1992

The pKa value of histidine-51 residue was determined by the pH dependency of contents of NADH bound to the active site in the orse liver alcohol dehydrogenase and % inactivation with diethyl pyrocarbonate treatment of the enzyme. The pKa for His-51 was -7.15 in the ternary complex and -6.7 in the enzyme itself.

• Journal of Physiology & Pathology in Korean Medicine
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• v.18 no.6
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• pp.1723-1727
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• 2004

We investigated the activity and stability of alcohol dehydrogenase from horse liver (HLADH) under the electric stimulation. The activity and stability of alcohol dehydrogenase depended on electric output voltaqe, stimulation time, pulse duration and pulse interval, and temperature. HLADH retained about 23% of its activity in buffer but 78% in 10% trehalose solution under electric stimulation with 10V, 10min, The stabilizing of enzymes against electric stimulation by stabilizing additives showed a great potential use of enzymes in biotechnology and medical engineering fields.

• Archives of Pharmacal Research
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• v.22 no.1
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• pp.13-17
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• 1999

The kinetic constants and the reaction mechanism of the K228G mutant horse liver alcohol dehyrogenase isoenzyme E (HLADH-E) were compared to the wild-type enzyme. All the Km and Ki constants of the mutant enzyme for NAD+, ethanol, acetaldehyde and NADH were larger than those of the wild-type enzyme. The dissociation constants for the NADH and $NAD^{+}$ (Kiq and Kia) were greatly increased by 130-and 460-fold, respectively. The product inhibition patterns suggested that the reaction mechanism of the mutant enzyme was changed to Random Bi Bi. These results could attribute to the increase in the dissociation rate of coenzyme with the substitution at Lys-228 residue.

• Archives of Pharmacal Research
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• v.18 no.2
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• pp.100-104
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• 1995

Lys-228 in horse liver alcohol dehydrogenase isoenzyme E(HLADH-E) was mutated to glycineby site-directed mutagenesis. The specific activity of the mutant enzyme was increased about 4-fold nad Michaelis constants for $NAD^+(K_a){\;}and{\;}NADH(K_q)$ increased by about 350-and 50-fold, respectively. The wild-type enzyme and K228TG mutant enzyme were treated with ethylacetimidate. Acetimidylation of the wild-type enzyme increased the activity about 10-fold, but the mutant enzyme ws little affected. These results confirm that Lys-228 residue plays an important role in the activity of the enzyme through forming the hydrogen bond with adenosine ribose of $NAD^+$.

• Journal of the Korean Chemical Society
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• v.45 no.1
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• pp.61-66
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• 2001

Stabilized Cross-linking Enzyme Crystals(CLEC) can be used as not only biocatalysts but also as enzyme sensors. PMS(Phenylmethyl Sulfate)was shown more efficience than any other electron mediator transfers toward HLADH(Horse Liver Alcohol Dehydrogenase)that were examined. NQS(naphtoquinonesulphonate), phenothiazine and ferrocene aldehyde had respectively just 52%, 37%, 35% electron transfer efficiency as compared to PMS . HLADH-CLEC was very stable toward elctron transfer mediators such as PMS, NQS and ferrocene aldehyde in which HLADH-solution was unstable.

• Bulletin of the Korean Chemical Society
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• v.9 no.1
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• pp.44-48
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• 1988

The feasibility of the reduction of nortricyclanone (1) as a chemical probe for testing the proposed radical mechanism for NAD-dependent horse liver alcohol dehydrogenase (HLADH) reactions has been examined using computer graphics modeling. The resutls of this study suggest that the radical ring-opening of this probe molecule may involve too substantial a geometry reorganization for the molecule to serve as a chemical probe in detecting the possible presence of the radical intermediates in the HLADH reactions. This result suggests that one should exercise caution in extrapolating results obtained from chemically based radical probes in the solution phase to the topologically constrained systems such as enzyme-substrate reactions.

• Bulletin of the Korean Chemical Society
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• v.11 no.6
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• pp.472-473
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• 1990

• YAKHAK HOEJI
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• v.36 no.5
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• pp.469-473
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• 1992

Yeast alcohol dehydrogenase (YADH) has an acidic residue that interacts with the 2'- and 3'-hydroxyl groups of the adenosine ribose of the $NAD^+$ coenzyme. The acidic residue of Asp-223 (according to horse liver alcohol dehydrogenase amino acid sequence) is supposed to determine the coenzyme specificity for $NAD^+$ rather than $NADP^+$. We mutated Asp-223 to leucine and the mutant YADH was expressed in yeast and characterized for the coenzyme specificity. The turnover numbers of mutant enzyme for $NAD^+$ and ethanol were decreased 3.5- and 4.8-fold compared to wild-type enzyme, respectively. Contrastively, catalytic specificity for $NADP^+$ was increased 13-fold. As a result, the mutant YADH also employed $NADP^+$ as a coenzyme.

• YAKHAK HOEJI
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• v.41 no.6
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• pp.756-764
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• 1997

Ile-224 in I269S mutant horse liver alcohol dehydrogenase isoenzyme S (HLADH-S) was mutated to serine by site-directed mutagenesis in order to study the role of the residue in c oenzyme binding to the enzyme. The specific activity of the I269S and I224S mutant enzyme to ethanol was increased 6-fold and all Michaelis constants($K_a,\;K_b,\;K_p,\;and\;K_q$,/TEX>) were larger than those for the wild-type and I269S enzyme. The substitution decreased the afffinity to coenzymes and increased the specific activity of the enzyme. The mutant enzyme showed the highest catalytic efficiency for octanol among the primary alcohols. But it didn`t have activities on retinoids and 5${\beta}$-cholanic acid-3-one. From these results, it was confirmed that the hydrophobic interaction of Ile-224 residue with coenzyme was related to coenzyme affinity in ADH reaction. The substitution also affected the substrate affinities to the enzyme.