A novel method for determining rate constants for dehydration of aldehyde hydrates

Rendina, Alan R.; Hermes, Jeffrey D.; Cleland, W. W.

doi:10.1021/bi00317a011

Cited by 29 publications

(29 citation statements)

References 34 publications

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“…Aldehydes and ketones can undergo rapid hydration, which can result in washout of 180 label and necessitate the use of a trap to prevent such washout. 119 The mechanism proposed in Scheme 5 also predicts that one atom of oxygen derived from 02 in the chase should be found in base propenal. We also recently developed a method to rapidly isolate base propenals from the reaction mixture.…”

Section: Proposed Mechanisms For Product Productionmentioning

confidence: 95%

“…The fact that no 180 from 1802 is observed in the base propenal in the experiment, for the mechanism in Scheme 6 to be valid, therefore requires 180 exchange from a precursor to the final product such as compound 8, Scheme 6. In analogy with CH3CHO, exchange is expected to occur rapidly from such an intermediate. 119 Rapid trapping of the proposed intermediate will, therefore, be required to establish the stoichiometric transfer of 180 from the chase 1802 into the base propenal precursor.…”

Section: Proposed Mechanisms For Product Productionmentioning

confidence: 99%

See 1 more Smart Citation

Mechanisms of bleomycin-induced DNA degradation

Stubbe¹,

Kozarich²

1987

Chem. Rev.

895

643

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Section: Proposed Mechanisms For Product Productionmentioning

confidence: 95%

Section: Proposed Mechanisms For Product Productionmentioning

confidence: 99%

Mechanisms of bleomycin-induced DNA degradation

Stubbe¹,

Kozarich²

1987

Chem. Rev.

895

643

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“…Carnitine acetyltransferase was substituted for acetate kinase, and ATP, phosphotransacetylase, and lactate dehydrogenase were omitted from the assay mixture. Glyoxylate (Rendina et al, 1984) and pyruvate (Bergmeyer, 1974) were assayed with lactate dehydrogenase and NADH.…”

Section: Methodsmentioning

confidence: 99%

Malate synthase: proof of a stepwise Claisen condensation using the double-isotope fractionation test

Clark

O'Keefe²,

Knowles³

1988

Biochemistry

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Although aldolase-catalyzed condensations proceed by stepwise mechanisms via the intermediacy of nucleophilic enol(ate)s or enamines, the mechanisms of those enzymes that catalyze Claisen-type condensations are unclear. The reaction pathway followed by an enzyme from this second group, malate synthase, has been studied by the double-isotope fractionation method to determine whether the reaction is stepwise or concerted. In agreement with earlier work, a deuterium kinetic isotope effect D(V/K) of 1.3 +/- 0.1 has been found when [2H3]acetyl-CoA is the substrate. The 13C isotope effect at the aldehydic carbon of glyoxylate has also been measured. For this determination, the malate product (containing the carbon of interest at C-2) was quantitatively transformed into a new sample of malate having the carbon of interest at C-4. This material was decarboxylated by malic enzyme to produce the appropriate CO2 for isotope ratio mass spectrometric analysis. The 13C isotope effect with [1H3]acetyl-CoA [that is, 13(V/K)H] is 1.0037 +/- 0.0004. By use of the known values of the intermolecular and intramolecular deuterium effects and of 13(V/K)H, the value of the 13C isotope effect when deuteriated [2H3]acetyl-CoA is the substrate [that is, 13(V/K)D] can be predicted for three possible mechanisms. If 13(V/K)H is a kinetic isotope effect and the reaction is concerted, the value of the 13C effect on deuteriation of acetyl-CoA will rise to 1.011; if 13(V/K)H is a kinetic isotope effect and the reaction is stepwise, the value of the 13C effect will fall to 1.0025; and if the 13C effect is an equilibrium isotope effect deriving from glyoxylate dehydration, the reaction is necessarily stepwise, and the value of 13(V/K)D will be 1.0037, unchanged from that of 13(V/K)H. Experimentally, the value of 13(V/K)D is 1.0037 +/- 0.0007, which requires that malate synthase follow a stepwise path. It is therefore clear that the two salient characteristics of enzymes that catalyze Claisen-like condensations, namely, the absence of enzyme-catalyzed proton exchange with solvent and the inversion of the configuration at the nucleophilic center, which had been suggestive of a concerted pathway, are not mechanistically diagnostic.

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“…This latter form is most likely the substrate for GO, as it is for LDH (49). In solution, the fraction of glyoxylate in the unhydrated form, determined from the equilibrium constant (K eq = 163), is 0.0038 (50,51). However, only the unhydrated form is visible in the crystal structure.…”

Section: Active Site Comparisonsmentioning

confidence: 99%

Active Site and Loop 4 Movements within Human Glycolate Oxidase: Implications for Substrate Specificity and Drug Design

2008

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Human glycolate oxidase (GO) catalyzes the FMN-dependent oxidation of glycolate to glyoxylate and glyoxylate to oxalate, a key metabolite in kidney stone formation. We report herein the structures of recombinant GO complexed with sulfate, glyoxylate, and an inhibitor, 4-carboxy-5-dodecylsulfanyl-1,2,3-triazole (CDST), determined by X-ray crystallography. In contrast to most alpha-hydroxy acid oxidases including spinach glycolate oxidase, a loop region, known as loop 4, is completely visible when the GO active site contains a small ligand. The lack of electron density for this loop in the GO-CDST complex, which mimics a large substrate, suggests that a disordered to ordered transition may occur with the binding of substrates. The conformational flexibility of Trp110 appears to be responsible for enabling GO to react with alpha-hydroxy acids of various chain lengths. Moreover, the movement of Trp110 disrupts a hydrogen-bonding network between Trp110, Leu191, Tyr134, and Tyr208. This loss of interactions is the first indication that active site movements are directly linked to changes in the conformation of loop 4. The kinetic parameters for the oxidation of glycolate, glyoxylate, and 2-hydroxy octanoate indicate that the oxidation of glycolate to glyoxylate is the primary reaction catalyzed by GO, while the oxidation of glyoxylate to oxalate is most likely not relevant under normal conditions. However, drugs that exploit the unique structural features of GO may ultimately prove to be useful for decreasing glycolate and glyoxylate levels in primary hyperoxaluria type 1 patients who have the inability to convert peroxisomal glyoxylate to glycine.

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A novel method for determining rate constants for dehydration of aldehyde hydrates

Cited by 29 publications

References 34 publications

Mechanisms of bleomycin-induced DNA degradation

Mechanisms of bleomycin-induced DNA degradation

Malate synthase: proof of a stepwise Claisen condensation using the double-isotope fractionation test

Active Site and Loop 4 Movements within Human Glycolate Oxidase: Implications for Substrate Specificity and Drug Design

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