Kinetics and native and modified liver alcohol dehydrogenase with coenzyme analogs: isomerization of enzyme-nicotinamide adenine dinucleotide complex

Coenzyme analogues with the adenosine ribose replaced with n-propyl, n-butyl, and n-pentyl groups; coenzyme analogues with the adenosine replaced with 3-(4-acetylanilino)propyl and 6-(4-acetylanilino)hexyl moieties; and nicotinamide mononucleotide, nicotinamide hypoxanthine dinucleotide, and 3-acety...

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Veröffentlicht in:Biochemistry (Easton) 1986-09, Vol.25 (19), p.5396-5402
Hauptverfasser: Plapp, Bryce V, Sogin, David C, Dworschack, Robert T, Bohlken, David P, Woenckhaus, Christoph, Jeck, Reinhard
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Sprache:eng
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Zusammenfassung:Coenzyme analogues with the adenosine ribose replaced with n-propyl, n-butyl, and n-pentyl groups; coenzyme analogues with the adenosine replaced with 3-(4-acetylanilino)propyl and 6-(4-acetylanilino)hexyl moieties; and nicotinamide mononucleotide, nicotinamide hypoxanthine dinucleotide, and 3-acetylpyridine adenine dinucleotide were used in steady-state kinetic studies with native and activated, amidinated enzymes. The Michaelis and inhibition constants increased up to 100-fold upon modification of coenzyme or enzyme. Turnover numbers with NAD+ and ethanol increased in some cases up to 10-fold due to increased rates of dissociation of enzyme-reduced coenzyme complexes. Rates of dissociation of oxidized coenzyme appeared to be mostly unaffected, but the values calculated (10-60 s-1) were significantly less than the turnover numbers with acetaldehyde and reduced coenzyme (20-900 s-1, at pH 8, 25 degrees C). Rates of association of coenzyme analogues also decreased up to 100-fold. When Lys-228 in the adenosine binding site was picolinimidylated, turnover numbers increased about 10-fold with NAD(H). Furthermore, the pH dependencies for association and dissociation of NAD+ and turnover number with NAD+ and ethanol showed the fastest rates above a pK value of 8.0. Turnover with NADH and acetaldehyde was fastest below a pK value of 8.1. These results can be explained by a mechanism in which isomerization of the enzyme-NAD+ complex (110 s-1) is partially rate limiting in turnover with NAD+ and ethanol (60 s-1) and is controlled by ionization of the hydrogen-bonded system that includes the water ligated to the catalytic zinc and the imidazole group of His-51.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi00367a008