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Origins of the high catalytic activity of human alcohol dehydrogenase 4 studied with horse liver A317C alcohol dehydrogenase.

ABSTRACT: The turnover numbers and other kinetic constants for human alcohol dehydrogenase (ADH) 4 ("stomach" isoenzyme) are substantially larger (10-100-fold) than those for human class I and horse liver alcohol dehydrogenases. Comparison of the primary amino acid sequences (69% identity) and tertiary structures of these enzymes led to the suggestion that residue 317, which makes a hydrogen bond with the nicotinamide amide nitrogen of the coenzyme, may account for these differences. Ala-317 in the class I enzymes is substituted with Cys in human ADH4, and locally different conformations of the peptide backbones could affect coenzyme binding. This hypothesis was tested by making the A317C substitution in horse liver ADH1E and comparisons to the wild-type ADH1E. The steady-state kinetic constants for the oxidation of benzyl alcohol and the reduction of benzaldehyde catalyzed by the A317C enzyme were very similar (up to about 2-fold differences) to those for the wild-type enzyme. Transient kinetics showed that the rate constants for binding of NAD(+) and NADH were also similar. Transient reaction data were fitted to the full Ordered Bi Bi mechanism and showed that the rate constants for hydride transfer decreased by about 2.8-fold with the A317C substitution. The structure of A317C ADH1E complexed with NAD(+) and 2,3,4,5,6-pentafluorobenzyl alcohol at 1.2 ? resolution is essentially identical to the structure of the wild-type enzyme, except near residue 317 where the additional sulfhydryl group displaces a water molecule that is present in the wild-type enzyme. ADH is adaptable and can tolerate internal substitutions, but the protein dynamics apparently are affected, as reflected in rates of hydride transfer. The A317C substitution is not solely responsible for the larger kinetic constants in human ADH4; thus, the differences in catalytic activity must arise from one or more of the other hundred substitutions in the enzyme.

SUBMITTER: Herdendorf TJ

PROVIDER: S-EPMC3103598 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

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Origins of the high catalytic activity of human alcohol dehydrogenase 4 studied with horse liver A317C alcohol dehydrogenase.

Herdendorf Timothy J TJ Plapp Bryce V BV

Chemico-biological interactions 20101222 1-3

The turnover numbers and other kinetic constants for human alcohol dehydrogenase (ADH) 4 ("stomach" isoenzyme) are substantially larger (10-100-fold) than those for human class I and horse liver alcohol dehydrogenases. Comparison of the primary amino acid sequences (69% identity) and tertiary structures of these enzymes led to the suggestion that residue 317, which makes a hydrogen bond with the nicotinamide amide nitrogen of the coenzyme, may account for these differences. Ala-317 in the class ...[more]

PMID: 21184752

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Project description:1. The inactivation of horse liver alcohol dehydrogenase by pyridoxal 5'-phosphate in phosphate buffer, pH8, at 10 degrees C was investigated. Activity declines to a minimum value determined by the pyridoxal 5'-phosphate concentration. The maximum inactivation in a single treatment is 75%. This limit appears to be set by the ratio of the first-order rate constants for interconversion of inactive covalently modified enzyme and a readily dissociable non-covalent enzyme-modifier complex. 2. Reactivation was virtually complete on 150-fold dilution: first-order analysis yielded an estimate of the rate constant (0.164min-1), which was then used in the kinetic analysis of the forward inactivation reaction. This provided estimates for the rate constant for conversion of non-covalent complex into inactive enzyme (0.465 min-1) and the dissociation constant of the non-covalent complex (2.8 mM). From the two first-order constants, the minimum attainable activity in a single cycle of treatment may be calculated as 24.5%, very close to the observed value. 3. Successive cycles of modification followed by reduction with NaBH4 each decreased activity by the same fraction, so that three cycles with 3.6 mM-pyridoxal 5'-phosphate decreased specific activity to about 1% of the original value. The absorption spectrum of the enzyme thus treated indicated incorporation of 2-3 mol of pyridoxal 5'-phosphate per mol of subunit, covalently bonded to lysine residues. 4. NAD+ and NADH protected the enzyme completely against inactivation by pyridoxal 5'-phosphate, but ethanol and acetaldehyde were without effect. 5. Pyridoxal 5'-phosphate used as an inhibitor in steady-state experiments, rather than as an inactivator, was non-competitive with respect to both NADH and acetaldehyde. 6. The partially modified enzyme (74% inactive) showed unaltered apparent Km values for NAD+ and ethanol, indicating that modified enzyme is completely inactive, and that the residual activity is due to enzyme that has not been covalently modified. 7. Activation by methylation with formaldehyde was confirmed, but this treatment does not prevent subsequent inactivation with pyridoxal 5'-phosphate. Presumably different lysine residues are involved. 8. It is likely that the essential lysine residue modified by pyridoxal 5'-phosphate is involved either in binding the coenzymes or in the catalytic step. 9. Less detailed studies of yeast alcohol dehydrogenase suggest that this enzyme also possesses an essential lysine residue.

Project description:The substrate specificities of alcohol dehydrogenases (ADH) are of continuing interest for understanding the physiological functions of these enzymes. Ser-48 and Phe-93 have been identified as important residues in the substrate binding sites of ADHs, but more comprehensive structural and kinetic studies are required. The S48T substitution in horse ADH1E has small effects on kinetic constants and catalytic efficiency (V/Km) with ethanol, but decreases activity with benzyl alcohol and affinity for 2,2,2-trifluoroethanol (TFE) and 2,3,4,5,6-pentafluorobenzyl alcohol (PFB). Nevertheless, atomic resolution crystal structures of the S48T enzyme complexed with NAD+ and TFE or PFB are very similar to the structures for the wild-type enzyme. (The S48A substitution greatly diminishes catalytic activity.) The F93A substitution significantly decreases catalytic efficiency (V/Km) for ethanol and acetaldehyde while increasing activity for larger secondary alcohols and the enantioselectivity for the R-isomer relative to the S-isomer of 2-alcohols. The doubly substituted S48T/F93A enzyme has kinetic constants for primary and secondary alcohols similar to those for the F93A enzyme, but the effect of the S48T substitution is to decrease V/Km for (S)-2-alcohols without changing V/Km for (R)-2-alcohols. Thus, the S48T/F93A substitutions invert the enantioselectivity for alcohol oxidation, increasing the R/S ratio by 10, 590, and 200-fold for 2-butanol, 2-octanol, and sec-phenethyl alcohol, respectively. Transient kinetic studies and simulations of the ordered bi bi mechanism for the oxidation of the 2-butanols by the S48T/F93A ADH show that the rate of hydride transfer is increased about 7-fold for both isomers (relative to wild-type enzyme) and that the inversion of enantioselectivity is due to more productive binding for (R)-2-butanol than for (S)-2-butanol in the ternary complex. Molecular modeling suggests that both of the sec-phenethyl alcohols could bind to the enzyme and that dynamics must affect the rates of catalysis.

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Origins of the high catalytic activity of human alcohol dehydrogenase 4 studied with horse liver A317C alcohol dehydrogenase.

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Origins of the high catalytic activity of human alcohol dehydrogenase 4 studied with horse liver A317C alcohol dehydrogenase.

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