Mechanism of Action of Coenzyme B₁₂ : I. Mechanism of Action of Coenzyme B₁₂. Hydrogen Transfer in the Isomerization of β-Methylaspartate to Glutamate. II. Kinetic Studies of 3-Fluoro-1,2-Propanediol, a New Substance for Dioldehydrase. III. Reaction of 5'-Deoxyinosylcobalamen with Propanediol Dehydrase. IV. Adenosine Deaminase

Author: Eagar, Robert Gouldman

Year: 1974

Degree: Dissertation (Ph.D.)

Advisor: Richards, John H.

Committee Member: Unknown, Unknown

Option: Chemistry

DOI: 10.7907/WKKX-RB26

Abstract

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PART I. Use of a mixture of non- and tetradeutero-beta-methylaspartate with coenzyme B12 dependent beta-methylaspartate-glutamate mutase has shown that the hydrogen that migrates becomes one of three equivalent hydrogens during the reaction. Kinetic isotope effects suggest that cleavage of the bond in the substrate from carbon to that hydrogen which migrates is an important component of the rate determining step. The evidence also supports the existence of an intermediate which can partition with similar probabilities to beta-methylaspartate or to glutamate. Some possible mechanistic implications of these findings are discussed.

PART II. The mechanism and the kinetics of the action of diol dehydrase on 3-fluoro-1,2-propanediol, a new substrate for diol dehydrase, were examined. The results of this study lead to a reexamination of the kinetic behavior of the natural substrate, 1,2-propanediol, with diol dehydrase. The [...] values of (S)-, (RS)-, and (R)-3-fluoro-1,2-propanediol were found to be 340[...], 128[...], and 104[...] respectively, compared to 368[...], 250[...], and 191[...], for the corresponding isomers of 1,2-propanediol. A modification of the existing analytical procedure for the determination of aldehyde was necessary in order to measure the values of Km for these substrates. Km values for (S)-, (RS)-, and (R)-3-fluoro-1,2-propanediol were found to be [...], [...], and [...] respectively. For (R)-, (RS)-, and (S)-1,2-propanediol, Km values were measured at [...], [...], and [...] respectively.

These results show that the (R)- and (S)- isomers of both substrates exhibit simple competitive behavior for the active site in the racemic mixture, and that both the (R)- and (S)- isomers of the substrate pass through a similar transition state in the enzyme catalyzed conversion to aldehyde.

PART III. The reactivity of 5'-deoxyinosylcobalamin in the propanediol dehydrase reaction was examined. This coenzyme B12 analogue showed no coenzyme activity in the diol dehydrase reaction. 5'-Deoxyinosylcobalamin was found to inhibit the activity of coenzyme B12 although it bound less efficiently than the natural coenzyme by a factor of 100. Previously reported experiments regarding the release of 5'-deoxyinosine by this system were shown to be inconclusive.

PART IV. The kinetic behavior of 5'-deoxyadenosine in the adenosine deaminase reaction has been examined. The low catalytic rate and inefficient binding observed for this substrate suggest that the 5'-hydroxyl is very important to both catalysis and binding of ribosyladenines. In spite of the low catalytic rate and the poor binding, adenosine deaminase was used to effectively synthesize 5'-deoxyinosine from 5'-deoxyadenosine.

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