The Mechanism of Pentose Phosphate Isomerization and Epimerization Studied with T2O and H2O18

McDonough, Michele; Wood, William F.

doi:10.1016/s0021-9258(18)64152-3

Cited by 39 publications

(9 citation statements)

References 36 publications

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“…Here no exchange of the hydrogens of dihydroxyacetone occurs; i.e., the proton removed from the dihydroxyacetone ketimine is shielded from exchange with bulk solvent by the protein (Tsolas and Horecker, 1972). The absence of exchange is also seen in a variety of epimerases such as ribulose-5phosphate 4-epimerase (McDonough and Wood, 1960), and lactate racemase Dennis, 1965, 1966).…”

Section: Discussionmentioning

confidence: 99%

Rabbit muscle aldolase catalyzed proton exchange of hydroxyacetone phosphate with solvent

Pratt¹

1977

Biochemistry

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Rabbit muscle aldolase catalyzes the exchange with solvent of all three methyl hydrogens of hydroxyacetone phosphate. Under saturating conditions, rates of the following processes have been measured: deuteration of hydroxyacetone phosphate in 2H2O (by an NMR method), tritiation of hydroxyacetone phosphate in H2O and 2H2O, and detritiation of tritiated hydroxyacetone phosphate in H2O and 2H2O. It is clear from these measurements (1) that there is no primary kinetic isotope effect and hence that hydrogen abstraction is not rate determining to the exchange and (2) that only one (as the closest integer) methyl hydrogen exchanges per turnover. The argument is made that these observations are mutually exclusive in terms of the accepted aldolase mechanism in the absence of further restrictions imposed by the enzyme. Possible restrictions are discussed.

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Section: Discussionmentioning

confidence: 99%

Rabbit muscle aldolase catalyzed proton exchange of hydroxyacetone phosphate with solvent

Pratt¹

1977

Biochemistry

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“…170-173 Solvent-derived 3 H or 18 O are not incorporated into the products and [4-3 H]-xylulose-5phosphate does not lose its label during epimerization. 170 In addition, no primary kinetic isotope effect was observed during the epimerization of [4-3 H]-xylulose-5-phosphate, demonstrating that cleavage of the C-4-H bond is not a rate determining step. These observations rule out all the mechanisms previously discussed in this review.…”

Section: Cofactor Independent Epimerasesmentioning

confidence: 97%

“…9 This mechanism is supported by the observation that solvent-derived deuterium is incorporated at C-3. 10 Moreover, a primary kinetic isotope effect has been observed in the epimerization of [3-2 H]--ribulose 5-phosphate. 11 Two potential strategies can be employed in the deprotonation/reprotonation event-a "one-base" mechanism or a "two-base" mechanism.…”

Section: Epimerases Acting On Free Sugarsmentioning

confidence: 99%

Mechanistic aspects of enzymatic carbohydrate epimerization

Samuel

Tanner

2002

Nat. Prod. Rep.

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Carbohydrate epimerases are enzymes that catalyze an inversion of stereochemistry at a stereogenic center in a sugar. This review focuses on the mechanistic strategies that are employed by this diverse family of enzymes, and is divided into the following sections: deprotonation/reprotonation, transient oxidation, cofactor independent, and mutarotation. The review is comprehensive up to Oct. 2001 and contains 216 references.

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“…Furthermore, the enzyme has been shown not to use an NAD + cofactor during catalysis (2), and therefore does not employ an oxidation/reduction mechanism analogous to that of UDP-galactose 4-epimerase (9,10). Other studies have shown that the reaction proceeds without the incorporation of solvent-derived oxygen or hydrogen atoms and that there is no primary kinetic isotope effect in the epimerization of either D-[4-3 H]Xu5P or L-[4-2 H]Ru5P (11)(12)(13). Two mechanisms have been proposed to explain these observations (6).…”

mentioning

confidence: 98%

Epimerization via Carbon−Carbon Bond Cleavage. l-Ribulose-5-phosphate 4-Epimerase as a Masked Class II Aldolase

Johnson

Tanner

1998

Biochemistry

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Studies indicating that the E. coli L-ribulose-5-phosphate 4-epimerase employs an "aldolase-like" mechanism are reported. This NAD+-independent enzyme epimerizes a stereocenter that does not bear an acidic proton and therefore it cannot utilize a simple deprotonation-reprotonation mechanism. Sequence similarities between the epimerase and the class II l-fuculose-1-phosphate aldolase suggest that the two may be evolutionarily related and that the epimerization may occur via carbon-carbon bond cleavage and re-formation. Conserved residues thought to provide the metal ion ligands of the epimerase have been modified using site-directed mutagenesis. The resulting mutants show low kcat values in addition to a reduced affinity for Zn2+. These observations serve to establish that there is a structural link between between the active site geometry of the epimerase and the aldolase. In addition, the H97N mutant was found to catalyze the condensation of dihydroxyacetone and glycolaldehyde phosphate to produce a mixture of L-ribulose-5-phosphate and D-xylulose-5-phosphate. This observation of aldolase activity establishes that the epimerase active site is capable of promoting carbon-carbon bond cleavage. Furthermore, glycolaldehyde phosphate was shown to be a competitive inhibitor of the mutant enzyme (KI = 0.37 mM) but not of the wild-type enzyme. The mutation apparently causes the epimerase to become "leaky" and enables it to bind/generate the normal reaction intermediates from the unbound aldol cleavage products.

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The Mechanism of Pentose Phosphate Isomerization and Epimerization Studied with T2O and H2O18

Cited by 39 publications

References 36 publications

Rabbit muscle aldolase catalyzed proton exchange of hydroxyacetone phosphate with solvent

Rabbit muscle aldolase catalyzed proton exchange of hydroxyacetone phosphate with solvent

Mechanistic aspects of enzymatic carbohydrate epimerization

Epimerization via Carbon−Carbon Bond Cleavage. l-Ribulose-5-phosphate 4-Epimerase as a Masked Class II Aldolase

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