13137-52-5Relevant articles and documents
A direct spectropolarimetric assay of arabinose 5-phosphate isomerase
Kijek, Todd M.,Bozue, Joel A.,Panchal, Rekha G.,Litosh, Vladislav A.,Woodard, Ronald W.,Ahmed, S. Ashraf
, (2021)
Arabinose 5-phosphate isomerase (API) catalyzes the reversible isomerization of Ribulose 5-phosphate (Ru5P) to Arabinose 5-Phosphate (Ar5P) for the production of 3-deoxy-2-octulosonic acid 8-phosphate (KDO), a component of bacterial lipopolysaccharide (LPS) of gram-negative bacteria. API is an attractive target for therapeutic development against gram-negative bacterial pathogens. The current assay method of API activity utilizes a general reaction for keto sugar determination in a secondary, 3-h color development reaction with 25 N sulfuric acid which poses hazard to both personnel and instrumentation. We therefore aimed to develop a more user friendly assay of the enzyme. Since Ru5P absorbs in the UV region and contains at least 2 chiral centers, it can be expected to display circular dichroism (CD). A wavelength scan revealed indeed Ru5P displays a pronounced negative ellipticity of 30,560 mDeg M?1cm?1 at 279 nm in Tris buffer pH 9.1 but Ar5P does not have any CD. API enzymatic reactions were monitored directly and continuously in real time by following the disappearance of CD from the Ru5P substrate, or by the appearance of CD from Ar5P substrate. The CD signal at this wavelength was not affected by absorption of the enzyme protein or of small molecules, or turbidity of the solution. Common additives in protein and enzyme reaction mixtures such as detergents, metals, and 5% dimethylsulfoxide did not interfere with the CD signal. Assay reactions of 1–3 min consistently yielded reproducible results. Introduction of accessories in a spectropolarimeter will easily adapt this assay to high throughput format for screening thousands of small molecules as inhibitor candidates of API.
Lipmann
, p. 588 (1936)
Reversible and in Situ Formation of Organic Arsenates and Vanadates as Organic Phosphate Mimics in Enzymatic Reactions: Mechanistic Investigation of Aldol Reactions and Synthetic Applications
Drueckhammer, Dale G.,Durrwachter, J. Robert,Pederson, Richard L.,Crans, Debbie C.,Daniels, Lacy,Wong, Chi-Huey
, p. 70 - 77 (2007/10/02)
A synthetic strategy is developed that uses organic phosphate utilizing enzymes as catalysts and a mixture of an organic alcohol and inorganic arsenate or vanadate to replace the organic phosphate substrate.In this process, inorganic arsenate or vanadate reacts with the alcohol reversibly in situ to form a mixture of esters, one of which is accepted by the enzyme as a substrate.Examples of the utility of this approach are demonstrated in enzymatic aldol condensations catalyzed by fructose-1,6-diphosphate aldolase, fuculose-1-phosphate aldolase, and rhamnulose-1-phosphate aldolase with a mixture of dihydroxyacetone and inorganic arsenate as substrate.Several uncommon sugars and deoxy sugars are prepared on 5-17-mmol scales.Mechanistic studies on an aldol reaction indicate that the redox reaction between dihydroxyacetone and inorganic vanadate prohibits the use of such a mixture to replace dihydroxyacetone phosphate in enzymatic aldol condensations.
Anomerization of Furanose Sugars and Sugar Phosphates
Pierce, John,Serianni, Anthony S.,Barker, Robert
, p. 2448 - 2456 (2007/10/02)
Thermodynamic and kinetic parameters for the ring-opening and -closing reactions of several aldo- and ketofuanoses and their phosphate esters have been determined by NMR line-width and saturation-transfer methods.Cyclic forms interconvert via a single, acyclic carbonyl form under either acid or base catalysis.Ring-opening rates do not correlate with thermodynamic stability of the rings.For aldofuranose phosphates, α anomers open faster than β anomers; for ketofuranose phosphates the converse is observed.Intramolecular catalysis of anomerization by the phosphate group of sugar phosphates is documented.Biological and mechanistic implications of the observed kinetics are discussed.
