Active-site cobalt(II)-substituted horse liver alcohol dehydrogenase: characterization of intermediates in the oxidation and reduction processes as a function of pH

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Abstract
Substitution of Co(II) for the catalytic site Zn(II) of horse liver alcohol dehydrogenase (LADH) yields an active enzyme derivative, CoIIE, with characteristic Co(II) charge-tranfer and d-d electronic transitions that are sensitive to the events which take place during catalysis [Koerber, S. C., MacGibbon, A. K. H., Dietrich, H., Zeppezauer, M., and Dunn, M. F. (1983) Biochemistry 22, 3424-3431]. In this study, UV-visible spectroscopy and rapid-scanning stopped-flow (RSSF) kinetic methods are used to detect and identify intermediates in the LADH catalytic mechanism. In the presence of the inhibitor isobutyramide, the pre-steady-state phase of alcohol (RCH2OH) oxidation at pH above 7 is characterized by the formation and decay of an intermediate with .lambda.max = 570, 640, and 672 nm for both aromatic and aliphatic alcohols (benzyl alcohl, p-nitrobenzyl alcohol, anisyl alcohol, ethanol, and methanol). By comparison with the spectrum of the stable ternary complex formed with oxidized nicotinamide adenine dinucleotide (NAD+) and 2,2'',2"-trifluoroethoxide ion (TFE-), CoIIE(NAD+,TFE-), the intermediate which forms is proposed to be the alkoxide ion (RCH2O-) complex, CoIIE(NAD+,RCH2O-). The timing of reduced nicotinamide adenine dinucleotide (NADH) formation indicates that intermediate decay is limited by the interconversion of ternary complexes, i.e., CoIIE(NAD+,RCH2O-) .dblarw. CoIIE(NADH,RCHO). From competition experiments, we inter that, at pH values below 5, NAD+ and alcohol form a CoIIE(NAD+,RCH2OH) ternary complex. RSSF studies carried out as a function of pH indicate tht the apparent pKa values for the ionization of alcohol within the ternary complex, i.e., CoIIE(NAD+,RCH2OH) .dblarw. CoIIE(NAD+,RCH2O-) + H+, fall in the range 5-7.5. Using pyrazole as the dead-end inhibitor, we find that the single-turnover time courses for the reduction of benzaldehyde, p-nitrobenzaldehyde, anisaldehyde, and acetaldehyde at pH above 7 all show evidence for the formation and decay of of an intermediate. Via spectral comparisons with CoIIE-(NAD+,TFE-) and with the intermediate formed during alcohol oxidation, we identify the intermediate as the same CoIIE(NAD+,RCH2O-) ternary complex detected during alcohol oxidation.
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