The Mechanism of Action of Adenosylcobalamin: I. 3-Fluoro-1, 2-Propanediol Dehydrase - Mechanistic Implications. II. Glycerol and Other Substrate Analogues as Substrates and Inactivators for Propanediol Dehydrase. Kinetics, Stereospecificity and Mechanism. III. The Involvement of a 5'-Deoxyadenosine Intermediate Containing Three Equivalent Hydrogens in the Mechanism of Glycerol Inactivation of Propanediol Dehydrase Holoenzyme. IV. Hydrogen Transfer from Enzyme-Bound Adenosylcobalamin as a Partial Rate Limiting Step in Catalysis

Author: Bachovchin, William Walter

Year: 1977

Degree: Dissertation (Ph.D.)

Advisor: Richards, John H.

Committee Member: Unknown, Unknown

Option: Chemistry

DOI: 10.7907/8fy8-dk59

Abstract

Part I

3-Fluoro-1,2-propanediol has been found to be a substrate for propanediol dehydrase and has very similar binding and catalytic constants compared to the natural substrate. The only isolable products of the reaction are acrolein and inorganic fluoride; with 3-fluoro-3,3-dideuterio- 1,2-propanediol as substrate, only 3,3-dideuterioacrolein is obtained. These results indicate that the primary product of the reaction is 3-fluoropropionaldehyde which spontaneously loses hydrogen fluoride to yield acrolein. The similar kinetic parameters for the fluorinated as compared to the normal substrate suggest that significant charge does not develop on the fluorinated or, by implication, the natural substrate during any rate-limiting steps of the reaction. These results support a radical, as contrasted to an ionic pathway for reactions involving adenosylcobalamin and diol dehydrase.

Part II

A number of vicinal diols were found to react with dioldehydrase, typically resulting in the conversion of enzyme-bound adenosylcobalamin to cob(II)alamin and formation of aldehyde or ketone derived from substrate. Moreover all are capable of effecting the irreversible inactivation of the enzyme. The kinetics and mechanism of product formation and inactivation were investigated.

Glycerol, found to be a very good substrate for diol dehydrase as well as a potent inactivator, atypically, did not induce cob(II)alamin formation to any detectable extent. With glycerol the inactivation process was accompanied by conversion of enzyme-bound adenosylcobalamin to an alkyl or thiol cobalamin, probably by substitution of an amino acid side chain near the active site for the 5'-deoxy-5'-adenosyl ligand on the cobalamin.

The inactivation reaction with glycerol as the inactivator exhibits a deuterium isotope effect of 14, strongly implicating hydrogen transfer as an important step in the mechanism of inactivation. The isotope effect of the rate of product formation was found to be 8.0.

Experiments with isotopically substituted glycerols indicate that dioldehydrase distinguishes between "R" and "S" binding conformations, the enzyme-"R"-glycerol complex being predominately responsible for the product-forming reaction while the enzyme-"S"-glycerol complex results primarily in the inactivation reaction. Mechanistic implications are discussed.

A method for removing enzyme-bound OH-Cbl that is non- destructive to the enzyme and a technique for measuring the binding constants of (R)-and (S)-1,2-propanediol are presented.

Part III

The kinetics of inactivation of adenosylcobalamin dependent propanediol dehydrase by glycerol, (RS)-1,1-dideuterioglycerol, (R)-1,1-dideuterioglycerol, and perdeuterioglycerol in the presence of 1,2-propanediol and 1,1-dideuterio-1,2-propanediol were investigated. The results (lead to the conclusion) that hydrogen (or deuterium) located on C-1 of 1,2-propanediol can participate in the inactivation-reaction and contribute to the expression of an isotope effect on the inactivation rate constant. The mechanism by which this occurs must involve the cofactor as intermediate hydrogen carrier, presumably in the form of 5'-deoxyadenosine. Moreover, the results can be quantitatively accounted for by a mechanism involving transfer of hydrogen from an intermediate containing three equivalent hydrogens, such as 5'-deoxyadeno-sine, as the rate-determining step in the inactivation pathway.

When dioldehydrase holoenzyme is inactivated with 1-3H-glycerol, 5'-deoxyadenosine enriched in tritium by a factor of 2.1 over that in glycerol can be isolated from the reaction mixture.

Part IV

The rate of catalysis by the adenosylcobalamin dependent enzyme diodehydrase was determined as a function of the relative amounts of 1,1-dideuterio-1,2-propanediol and 1,2-propanediol present in the reaction mixture. The rate was found to decrease sharply at low mole fractions of 1,1-dideuterio-1,2-propanediol and slowly approach the rate observed 1,1-dideuterio-1,2 -propanediol at large mole fractions of 1,1-dideuterio-1,2-propanediol. We interpret this to indicate that hydrogen transfer from enzyme-bound adenosylcobalamin is at least partially rate limiting in catalysis.

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