Deprotonation of the Horse Liver Alcohol Dehydrogenase-NAD+ Complex Controls Formation of the Ternary Complexes

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• 作者：Elena G. Kovaleva and Bryce V. Plapp
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：September 27, 2005
• 年：2005
• 卷：44
• 期：38
• 页码：12797 - 12808
• 全文大小：307K
• 年卷期：v.44,no.38(September 27, 2005)
• ISSN：1520-4995

文摘

Binding of NAD⁺ to wild-type horse liver alcohol dehydrogenase is strongly pH-dependentand is limited by a unimolecular step, which may be related to a conformational change of the enzyme-NAD⁺ complex. Deprotonation during binding of NAD⁺ and inhibitors that trap the enzyme-NAD⁺complex was examined by transient kinetics with pH indicators, and formation of complexes was monitoredby absorbance and protein fluorescence. Reactions with pyrazole and trifluoroethanol had biphasic protonrelease, whereas reaction with caprate showed proton release followed by proton uptake. Proton release(200-550 s^-1) is a common step that precedes binding of all inhibitors. At all pH values studied, the rateconstants for proton release or uptake matched those for formation of ternary complexes, and the mostsignificant quenching of protein fluorescence (or perturbation of adenine absorbance at 280 nm) wasobserved for enzyme species involved in deprotonation steps. Kinetic simulations of the combined transientdata for the multiple signals indicate that all inhibitors bind faster and tighter to the unprotonated enzyme-NAD⁺ complex, which has a pK of about 7.3. The results suggest that rate-limiting deprotonation of theenzyme-NAD⁺ complex is coupled to the conformational change and controls the formation of ternarycomplexes.