Rabbit muscle aldolase catalyzed proton exchange of hydroxyacetone phosphate with solvent

Pratt, Robertson

doi:10.1021/bi00637a008

Cited by 14 publications

(7 citation statements)

References 42 publications

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“…Thus, referring to Scheme , k −1 is large compared with k 2 , and the process that corresponds to k 1 / k −1 can be treated as a rapid pre‐equilibrium. Similar behavior has been observed by Pratt in the RMA‐catalyzed (RMA=rabbit‐muscle aldolase) deuteration of hydroxyacetone phosphate19 and by Westheimer in the acetoacetate decarboxylase‐catalyzed de‐deuteration reaction with [D 6 ]acetone 20. This model of a rapid pre‐equilibrium fits the kinetics of most of the ketone and aldehyde substrates shown in Tables 1–4.…”

Section: Resultssupporting

confidence: 80%

“…The lack of a large isotope effect, which would be expected if the formation of the enamine occurred in the rate determining step, probably reflects a nonsymmetrical transition state. Similar observations and interpretations have already been reported for RMA 19. It is remarkable that a catalytic antibody and highly evolved enzymes share the fine details of their catalytic mechanisms.…”

Section: Resultsmentioning

confidence: 99%

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Highly Efficient Antibody-Catalyzed Deuteration of Carbonyl Compounds

et al. 2002

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Antibody 38C2 efficiently catalyzes deuterium-exchange reactions at the alpha position of a variety of ketones and aldehydes, including substrates that have a variety of sensitive functional groups. In addition to the regio- and chemoselectivity of these reactions, the catalytic rates (kcat) and rate-enhancement values (kcat/kun) are among the highest values ever observed with catalytic antibodies. Comparison of the substrate range of the catalytic antibody with highly evolved aldolase enzymes, such as rabbit-muscle aldolase, highlights the much broader practical scope of the antibody, which accepts a wide range of substrates. The hydrogen-exchange reaction was used for calibration and mapping of the antibody active site. Isotope-exchange experiments with cycloheptanone reveal that the formation of the Schiff base species (as concluded from the 16O/18O exchange rate at the carbonyl oxygen) is much faster than the formation of the enamine intermediate (as concluded from the H/D exchange rate), and both steps are faster than the antibody-catalyzed aldol addition reaction.

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Section: Resultssupporting

confidence: 80%

Section: Resultsmentioning

confidence: 99%

Highly Efficient Antibody-Catalyzed Deuteration of Carbonyl Compounds

et al. 2002

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“…Mechanistic studies on FDP aldolase have shown that neither Schiff base formation/hydrolysis nor C–C bond formation/cleavage is rate limiting. Instead, the slowest steps for both FDP cleavage and synthesis are associated with product release. − …”

Section: Discussionmentioning

confidence: 99%

Optimization of Enzyme Mechanism along the Evolutionary Trajectory of a Computationally Designed (Retro-)Aldolase

2017

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De novo biocatalysts have been successfully generated by computational design and subsequent experimental optimization. Here, we examined the evolutionary history of the computationally designed (retro-)aldolase RA95. The modest activity of the starting enzyme was previously improved 10 5 -fold over many rounds of mutagenesis and screening to afford a proficient biocatalyst for enantioselective cleavage and synthesis of β-hydroxyketones. Using a set of representative RA95 variants, we probed individual steps in the multistep reaction pathway to determine which processes limit steady-state turnover and how mutations that accumulated along the evolutionary trajectory influenced the kinetic mechanism. We found that the overall rate-limiting step for aldol cleavage shifted from C−C bond scission (or an earlier step in the pathway) for the computational design to product release for the evolved enzymes. Specifically, interconversion of Schiff base and enamine intermediates, formed covalently between acetone and the catalytic lysine residue, was found to be the slowest step for the most active variants. A complex hydrogen bond network of four active site residues, which was installed in the late stages of laboratory evolution, apparently enhances lysine reactivity and facilitates efficient proton shuffling. This catalytic tetrad accounts for the tremendous rate acceleration observed for all steps of the mechanism, most notably Schiff base formation and hydrolysis. Comparison of our results with kinetic and structural studies on natural aldolases provides valuable feedback for computational enzyme design and laboratory evolution approaches alike.

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“…by a procedure similar to that reported for the synthesis of 1 -halo-3-hydroxyacetone phosphates (Silverman et al, 1975). Hydrogenolysis (Brown & Brown, 1966) of the washed intermediate dibenzyl ester yielded 440 mg (23% overall yield) of crude hydroxyacetone dihydrogen phosphate, which was purified by ion-exchange chromatography on a column of Dowex-1 (Cl) resin (Pratt, 1977). The product was quantitatively recovered from the column and contained 3% inorganic phosphate, as shown by phosphate determinations before and after ashing (Ames & Dubin, 1960), using a modification (Curthoys, 1966) of the method of Fiske & Subbarow (1925).…”

Section: Methodsmentioning

confidence: 99%