EC 5. Isomerases
EC 5.1 Racemases and Epimerases
EC 5.1.1 Acting on Amino Acids and Derivatives
EC 5.1.1.1 alanine racemase
EC 5.1.1.2 methionine racemase
EC 5.1.1.3 glutamate racemase
EC 5.1.1.4 proline racemase
EC 5.1.1.5 lysine racemase
EC 5.1.1.6 threonine racemase
EC 5.1.1.7 diaminopimelate epimerase
EC 5.1.1.8 4-hydroxyproline epimerase
EC 5.1.1.9 arginine racemase
EC 5.1.1.10 amino-acid racemase
EC 5.1.1.11 phenylalanine racemase (ATP-hydrolysing)
EC 5.1.1.12 ornithine racemase
EC 5.1.1.13 aspartate racemase
EC 5.1.1.14 nocardicin-A epimerase
EC 5.1.1.15 2-aminohexano-6-lactam racemase
EC 5.1.1.16 protein-serine epimerase
EC 5.1.1.17 isopenicillin-N epimerase
EC 5.1.1.18 serine racemase
EC 5.1.2 Acting on Hydroxy Acids and Derivatives
EC 5.1.2.1 lactate racemase
EC 5.1.2.2 mandelate racemase
EC 5.1.2.3 3-hydroxybutyryl-CoA epimerase
EC 5.1.2.4 acetoin racemase
EC 5.1.2.5 tartrate epimerase
EC 5.1.2.6 isocitrate epimerase
EC 5.1.3 Acting on Carbohydrates and Derivatives
EC 5.1.3.1 ribulose-phosphate 3-epimerase
EC 5.1.3.2 UDP-glucose 4-epimerase
EC 5.1.3.3 aldose 1-epimerase
EC 5.1.3.4 L-ribulose-5-phosphate 4-epimerase
EC 5.1.3.5 UDP-arabinose 4-epimerase
EC 5.1.3.6 UDP-glucuronate 4-epimerase
EC 5.1.3.7 UDP-N-acetylglucosamine 4-epimerase
EC 5.1.3.8 N-acylglucosamine 2-epimerase
EC 5.1.3.9 N-acylglucosamine-6-phosphate 2-epimerase
EC 5.1.3.10 CDP-paratose 2-epimerase
EC 5.1.3.11 cellobiose epimerase
EC 5.1.3.12 UDP-glucuronate 5′-epimerase
EC 5.1.3.13 dTDP-4-dehydrorhamnose 3,5-epimerase
EC 5.1.3.14 UDP-N-acetylglucosamine 2-epimerase
EC 5.1.3.15 glucose-6-phosphate 1-epimerase
EC 5.1.3.16 UDP-glucosamine 4-epimerase
EC 5.1.3.17 heparosan-N-sulfate-glucuronate 5-epimerase
EC 5.1.3.18 GDP-mannose 3,5-epimerase
EC 5.1.3.19 chondroitin-glucuronate 5-epimerase
EC 5.1.3.20 ADP-glyceromanno-heptose 6-epimerase
EC 5.1.3.21 maltose epimerase
EC 5.1.3.22 L-ribulose-5-phosphate 3-epimerase
EC 5.1.3.23 UDP-2,3-diacetamido-2,3-dideoxyglucuronic acid 2-epimerase
EC 5.1.99 Acting on Other Compounds
EC 5.1.99.1 methylmalonyl-CoA epimerase
EC 5.1.99.2 16-hydroxysteroid epimerase
EC 5.1.99.3 allantoin racemase
EC 5.1.99.4 α-methylacyl-CoA racemase
EC 5.1.99.5 hydantoin racemase
EC 5.2 cis-trans-Isomerases
EC 5.2.1.1 maleate isomerase
EC 5.2.1.2 maleylacetoacetate isomerase
EC 5.2.1.3 retinal isomerase
EC 5.2.1.4 maleylpyruvate isomerase
EC 5.2.1.5 linoleate isomerase
EC 5.2.1.6 furylfuramide isomerase
EC 5.2.1.7 retinol isomerase
EC 5.2.1.8 peptidylprolyl isomerase
EC 5.2.1.9 farnesol 2-isomerase
EC 5.2.1.10 2-chloro-4-carboxymethylenebut-2-en-1,4-olide isomerase
EC 5.2.1.11 deleted entry
EC 5.3 Intramolecular Oxidoreductases
EC 5.3.1 Interconverting Aldoses and Ketoses
EC 5.3.1.1 triose-phosphate isomerase
EC 5.3.1.2 deleted
EC 5.3.1.3 arabinose isomerase
EC 5.3.1.4 L-arabinose isomerase
EC 5.3.1.5 xylose isomerase
EC 5.3.1.6 ribose-5-phosphate isomerase
EC 5.3.1.7 mannose isomerase
EC 5.3.1.8 mannose-6-phosphate isomerase
EC 5.3.1.9 glucose-6-phosphate isomerase
EC 5.3.1.10 now EC 3.5.99.6
EC 5.3.1.11 deleted
EC 5.3.1.12 glucuronate isomerase
EC 5.3.1.13 arabinose-5-phosphate isomerase
EC 5.3.1.14 L-rhamnose isomerase
EC 5.3.1.15 D-lyxose ketol-isomerase
EC 5.3.1.16 1-(5-phosphoribosyl)-5-[(5-phosphoribosylamino)methylideneamino]imidazole-4-carboxamide isomerase
EC 5.3.1.17 4-deoxy-L-threo-5-hexosulose-uronate ketol-isomerase
EC 5.3.1.18 deleted
EC 5.3.1.19 now EC 2.6.1.16
EC 5.3.1.20 ribose isomerase
EC 5.3.1.21 corticosteroid side-chain-isomerase
EC 5.3.1.22 hydroxypyruvate isomerase
EC 5.3.1.23 5-methylthioribose-1-phosphate isomerase
EC 5.3.1.24 phosphoribosylanthranilate isomerase
EC 5.3.1.25 L-fucose isomerase
EC 5.3.1.26 galactose-6-phosphate isomerase
EC 5.3.1.27 6-phospho-3-hexuloisomerase
EC 5.3.1.28 D-sedoheptulose 7-phosphate isomerase
EC 5.3.2 Interconverting Keto- and Enol-Groups
EC 5.3.2.1 phenylpyruvate tautomerase
EC 5.3.2.2 oxaloacetate tautomerase
EC 5.3.3 Transposing C=C Bonds
EC 5.3.3.1 steroid Δ-isomerase
EC 5.3.3.2 isopentenyl-diphosphate Δ-isomerase
EC 5.3.3.3 vinylacetyl-CoA Δ-isomerase
EC 5.3.3.4 muconolactone Δ-isomerase
EC 5.3.3.5 cholestenol Δ-isomerase
EC 5.3.3.6 methylitaconate Δ-isomerase
EC 5.3.3.7 aconitate Δ-isomerase
EC 5.3.3.8 dodecenoyl-CoA Δ-isomerase
EC 5.3.3.9 prostaglandin-A1 .................More Read....
Prepared by
Nam Sun Wang
Department of Chemical & Biomolecular Engineering
University of Maryland
College Park, MD 20742-2111
ENCH485
The reaction between alpha-amino acid and ninhydrin involved in the development of color are described by the following five mechanistic steps:
alpha-amino acid + ninhydrin ---> reduced ninhydrin + alpha-amino acid + H2O alpha-amino acid + H2O ---> alpha-keto acid +NH3 alpha-keto acid + NH3 ---> aldehyde + CO2
Step (1) is an oxidative deamination reaction that removes two hydrogen from the alpha-amino acid to yield an alpha-imino acid. Simultaneously, the original ninhydrin is reduced and loses an oxygen atom with the formation of a water molecule. In Step (2), the NH group in the alpha-imino acid is rapidly hydrolyzed to form an alpha-keto acid with the production of an ammonia molecule. This alpha-keto acid further undergoes decarboxylation reaction of Step (3) under a heated condition to form an aldehyde that has one less carbon atom than the original amino acid. A carbon dioxide molecule is produced here. These first three steps produce the reduced ninhydrin and ammonia that are required for the production of color in the last two Steps (4) and (5). The overall reaction for the above reactions is simply (slightly inaccurately) expressed in Reaction (6) as follows:
alpha-amino acid + 2 ninhydrin ---> CO2 + aldehyde + final complex(BlUE) + 3H2O
In summary, ninhydrin, which is originally yellow, reacts with amino acid and turns deep purple. It is this purple color that is detected in this method.Ninhydrin will react with a free alpha-amino group, NH2-C-COOH. This group is contained in all amino acids, peptides, or proteins. Whereas, the decarboxylation reaction will proceed for a free amino acid, it will not happen for peptides and proteins. Thus, theoretically only amino acids will lead to the color development. However, one should always check out the possible interference from peptides and proteins by performing blank tests especially when such solutions are readily available. For example, one can simply add the ninhydrin reagent to a solution of only proteins and see if there is any color development. There is no excuse for failing to perform such a vital test when the sample mixture contains both proteins and amino acids. There are also reports that chemical compounds other than amino acids also yield positive results.
This test can be used routinely for the detection of glycine in the absence of other interfering species. Although this is a fast and sensitive test for the presence of alpha-amino acids, because of the nonselectivity, it cannot be used to analyze the relative individual contents of a mixture of different amino acids. Furthermore, the color intensity developed is dependent on the type of amino acid. Finally, it does not react with tertiary or aromatic amines.
Note that since ninhydrin is a strong oxidizing agent, proper caution should be exercised in handling this compound. It is especially potent at the elevated temperature under which the reaction is carried out. The ninhydrin reagent will stain the skin blue and cannot be immediately washed off completely if it comes in contact with the skin. However, as in any other stain on the skin, the color will gradually rub off after about a day.
Absorbance assays are fast and convenient, since no additional reagents or incubations are required. No protein standard need be prepared. The assay does not consume the protein. The relationship of absorbance to protein concentration is linear. Because different proteins and nucleic acids have widely varying absorption characteristics there may be considerable error, especially for unknowns or protein mixtures. Any non-protein component of the solution that absorbs ultraviolet light will intefere with the assay. Cell and tissue fractionation samples often contain insoluble or colored components that interfere. The most common use for the absorbance assay is to monitor fractions from chromatography columns, or any time a quick estimation is needed and error in protein concentration is not a concern. An absorbance assay is recommended for calibrating bovine serum albumin or other pure protein solutions for use as standards in other methods.
Concentration (mg/ml) = Absorbance at 280 nm divided by path length (cm.)
Pure protein of known absorbance coefficient. Use the following formula for a path length of 1 cm. Concentration is in mg/ml, %, or molarity depending on which type coefficient is used.
concentration = Absorbance at 280 nm divided by absorbance coefficient
To convert units, use these relationships:
Mg protein/ml = % protein divided by 10 = molarity divided by protein molecular weight
Unknowns with possible nucleic acid contamination. Use the following formula to estimate protein concentration:
Concentration (mg/ml) = (1.55 x A280) – 0.76 x A260)
Absorbance coefficients of some common protein standards: