Diol Dehydratase: Purification, Structural Characterization, and Mechanism of Action

Citation

McGee, Dennis Emmett (1983) Diol Dehydratase: Purification, Structural Characterization, and Mechanism of Action. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/xkz2-e227. https://resolver.caltech.edu/CaltechTHESIS:04082021-204133329

Abstract

Abstract to Chapter I

A new isolation procedure for propanediol dehydratase increases by a factor of about 16 the yield of enzyme obtainable from Klebsiella pneumoniae; the enzyme thus isolated has a specific activity of 95 ± 4 units/mg. The apoenzyme consists of four different subunits with molecular weights of 60 K, 51 K, 29 K, and 15 K daltons in the ratio of 2:1:2:2, respectively. In this new procedure, care was taken to prevent the partial proteolysis of the propanediol dehydratase which seems to occur in earlier procedures. The other novel aspect recognizes that the enzyme is associated with the cell membrane. Accordingly, after gentle sonication, the membrane fragments are separated from cytosol, and the enzyme is solubilized by extraction with buffers containing detergent. The amino acid compositions and N-terminal amino acid sequences for each of the subunits was also determined. From the amino acid compositions of the individual subunits, diol dehydratase appears to be a peripheral membrane protein.

Abstract to Chapter II

When diol dehydratase holoenzyme is inactivated by reaction with radioactive glycerol, one mole of glycerol appears to become tightly associated with each 250,000 daltons of the holoenzyme complex with a significant loss of tritium from C-2 being observed. However, denaturation of the inactivated complex releases the modified glycerol from the protein, indicating that the protein is not covalently modified by the inactivator. Similar experiments were carried out with radioactive isobutylene glycol, but due to the high level of nonspecific labeling, the results were not as definitive.

As described in Chapter I, former isolation procedures (Abeles, 1966; Poznanskaya et al., 1979) yielded enzyme which had been proteolysed. For this reason inactivation studies employing various deuterated derivatives of glycerol and isobutylene glycol, as well as a new class of inactivators represented by hydroxyacetone and dihydroxyacetone, were carried out with native enzyme to compare results from similar studies with proteolysed enzyme (Bachovchin et al., 1977; Moore, 1979). It was found that proteolysis had little effect on the constants associated with the glycerol inactivation, but an enormous effect on the constants describing the inactivation by isobutylene glycol.

The results of the radiolabeling studies and kinetic experiments are consistent with the formation of a secondary alkylcobalamin upon inactivation of diol dehydratase by glycerol. Kinetic evidence also suggests that the inactivation of diol dehydratase by isobutylene glycol occurs after the abstraction of hydrogen from C-1, but before the substrate rearranges.

Abstract to Chapter III

A reinvestigation of trace label experiments with native diol dehydratase isolated by the method of McGee and Richards (1981) (see Chapter I) has shown that the probability of net intramolecular transfer for tritium is 0.33 ± 0.02 as opposed to the value of about 0.03 obtained earlier (Frey et al., 1967a) with an enzyme preparation obtained by a different method. Our observed value of 0.33 is about 20 times larger than what one would predict on the basis of the mechanism for the migration of hydrogen given in the Introduction to this thesis. In contrast, tritium washout experiments, similar to those conducted by Essenberg et al. (1971), yielded a value of k_HH/k_HT = 29 ± 2 which is approximately the value predicted by Moore et al. (1979). Also, tritium washout experiments were carried out in such a way that, in addition to the C-5' hydrogens of adenosylcobalamin containing tritium, any other multiple-hydrogen reservoirs should have contained tritium as well. These experiments yielded identical results to those in which only the C-5' hydrogens contained reactable tritium; therefore, it appears that only the C-5' hydrogens of C-5' deoxyadenosine participate directly in catalysis.

Also, the tritium isotope effect on the first hydrogen transfer was determined to be 6.1 ± 0.5 by measuring the isotopic enrichment in unreacted [1-³H]-1,2-propanediol as a function of the extent of reaction. The results of our study suggest that the generally accepted pathway for catalysis, which proceeds through the C-5' deoxyadenosine hydrogen reservoir, constitutes about 95% of the catalytic events with unlabeled substrate; however, there appears to be an alternate catalytic pathway whose contribution to catalysis can be enhanced by isotopic substitution at C-1 of substrate.

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