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Correlation studies based on enzyme structure and kinetic results deduction of productive substrate orientation in the active site pocket of horse liver alcohol dehydrogenase

Dutler, H.; Branden C I.

Bioorganic Chemistry 10(1): 1-13

1981


ISSN/ISBN: 0045-2068
DOI: 10.1016/0045-2068(81)90038-9
Accession: 005061771

Kinetic information on the reduction of alkylated cyclohexanones catalyzed by horse liver alcohol dehydrogenase was correlated with the 3-dimensional structure of the enzyme. The substrates investigated were: 2-, 3- and 4-alkylcyclohexanones with alkyl groups methyl, ethyl, i-propyl and t-butyl. Kinetic studies establishing at which position of the cyclohexanone ring an alkyl group leads to fast, slow or no reduction and at which position an increase in the size of the alkyl group leads to a decrease of the rate of reduction, allows one to deduce at which position an alkyl group leads to favorable or unfavorable interactions with groups of the enzyme. On the basis of the X-ray structure of the enzyme and on plausible assumptions regarding the arrangement of the reacting atoms, models were built of the enzyme-NAD+-alkylcyclohexanol complexes formed during reduction. These models were analyzed with respect to favorable and unfavorable interactions. By changing the orientation of the cyclohexanol molecules in the complex it was possible to arrive at a structure in which the interactions observed in the model correlated extremely well with those deduced from kinetic analysis. As a result, a probable structure of the enzyme coenzyme substrate complex with productive substrate orientation was obtained. In this orientation the O2 of the substrate appears to be directly bound to the active-site Zn. The excellent correlation between the kinetic and the structural information demonstrates that the method of kinetically deducing the occurrence of interactions between groups of the substrate and the enzyme can be used to obtain information about the topography of the active site.

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