6o91

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== Publication Abstract from PubMed ==
== Publication Abstract from PubMed ==
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Primary and secondary kD/kT and kH/kT kinetic isotope effects have been studied as a probe of hydrogen tunneling in the oxidation of benzyl alcohol catalyzed by horse liver alcohol dehydrogenase (LADH). In the case of the wild-type enzyme, isotope effects at 25 degrees C do not clearly support hydrogen tunneling; this result is consistent with a reaction rate that is partially limited by the release of product benzaldehyde. The three-dimensional structure for LADH was used to design site-directed mutations in an effort to enhance the rate of the product release step and to "unmask" tunneling. Substitutions that increased the size of the alcohol binding pocket resulted in minor changes in isotope effects. By contrast, reduction in the size of the alcohol binding pocket through substitution at residues 57 and 93, which are in van der Waals contact with bound alcohol substrate, produced a clear demonstration of protium tunneling from the breakdown of the semiclassical relationship between kD/kT and kH/kT isotope effects. The temperature dependence of kD/kT isotope effects has also been pursued, leading to the conclusion that tunneling does, in fact, occur in the reaction catalyzed by wild-type LADH. Despite the unmasking of protium tunneling in site-directed mutants, substitutions that decrease the size of the alcohol pocket appear to result in less extensive tunneling in the hydride transfer. It is noteworthy that the mutant enzyme (Leu57--&gt;Phe), which shows the greatest evidence of tunneling, has the same catalytic efficiency (Vmax/Km) as the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
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Previous studies showed that the L57F and F93W alcohol dehydrogenases catalyze the oxidation of benzyl alcohol with some quantum mechanical hydrogen tunneling, whereas the V203A enzyme has diminished tunneling. Here, steady-state kinetics for the L57F and F93W enzymes were studied, and microscopic rate constants for the ordered bi-bi mechanism were estimated from simulations of transient kinetics for the S48T, F93A, S48T/F93A, F93W, and L57F enzymes. Catalytic efficiencies for benzyl alcohol oxidation (V1/EtKb) vary over a range of approximately 100-fold for the less active enzymes up to the L57F enzyme and are mostly associated with the binding of alcohol rather than the rate constants for hydride transfer. In contrast, catalytic efficiencies for benzaldehyde reduction (V2/EtKp) are approximately 500-fold higher for the L57F enzyme than for the less active enzymes and are mostly associated with the rate constants for hydride transfer. Atomic-resolution structures (1.1 A) for the F93W and L57F enzymes complexed with NAD(+) and 2,3,4,5,6-pentafluorobenzyl alcohol or 2,2,2-trifluoroethanol are almost identical to previous structures for the wild-type, S48T, and V203A enzymes. Least-squares refinement with SHELXL shows that the nicotinamide ring is slightly strained in all complexes and that the apparent donor-acceptor distances from C4N of NAD to C7 of pentafluorobenzyl alcohol range from 3.28 to 3.49 A (+/-0.02 A) and are not correlated with the rate constants for hydride transfer or hydrogen tunneling. How the substitutions affect the dynamics of reorganization during hydrogen transfer and the extent of tunneling remain to be determined.
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Unmasking of hydrogen tunneling in the horse liver alcohol dehydrogenase reaction by site-directed mutagenesis.,Bahnson BJ, Park DH, Kim K, Plapp BV, Klinman JP Biochemistry. 1993 Jun 1;32(21):5503-7. PMID:8504071<ref>PMID:8504071</ref>
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Substitutions of Amino Acid Residues in the Substrate Binding Site of Horse Liver Alcohol Dehydrogenase Have Small Effects on the Structures but Significantly Affect Catalysis of Hydrogen Transfer.,Kim K, Plapp BV Biochemistry. 2020 Feb 10. doi: 10.1021/acs.biochem.9b01074. PMID:31994873<ref>PMID:31994873</ref>
From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br>
From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br>

Revision as of 04:40, 13 February 2020

Horse liver L57F alcohol dehydrogenase complexed with NAD and pentafluorobenzyl alcohol

PDB ID 6o91

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