Mutarotation complex

Further experiments by Brown and particularly Henri were made with invertase. At that time the pH of the reactions was not controlled, mutarotation did not proceed to completion, and it is no longer possible to identify how much enzyme was used (Segal, 1959). Nevertheless, in a critical review of kinetic studies with invertase, Henri concluded (1903) that the rate of reaction was proportional to the amount of enzyme. He also stated that the equilibrium of the enzyme-catalyzed reaction was unaffected by the presence of the catalyst, whose concentration remained unchanged even after 10 hours of activity. When the concentration of the substrate [S] was sufficiently high the velocity became independent of [S]. Henri derived an equation relating the observed initial velocity of the reaction, Vq, to the initial concentration of the substrate, [S0], the equilibrium constant for the formation of an enzyme-substrate complex, Ks, and the rate of formation of the products, ky... 

In aqueous solution the free nitriles mutarotate in a complex manner that indicates the existence of at least three compounds in the equilibrated solutions. This reaction has been interpreted as a reversible transformation of the nitriles into other substances. 

The original publication by Sweeley and coworkers5 was concerned with the separation of a wide range of carbohydrates, from mono- to tetra-saccharides. Most of the subsequent publications have considered the quantitative analysis of mixtures of varied complexity, although two studies have demonstrated the separation of the protium from the deuterium fonns of monosaccharides.200,201 The study of mutarotational equilibria by gas-liquid chromatography has been discussed in Section IV (see p. 38). 

It is easy to see from Equation 8 why —NH3+ ion does not catalyze the mutarotation The positively charged ion cannot extract the proton from the hydroxyl group on carbon 1. When no NaOH is added, in the presence of 0.0114M Cd(N03)2, the rate constant of mutarotation is 0.0122, practically the same as in the absence of the metal. This is as expected, since no glucosamine complex is present. 

The majority of the many methods used to study the composition of equilibrium solutions of carbohydrates examine the mixture without separating the individual components. With the discovery that the anomeric forms of sugars could be readily separated by gas chromatography of their tri-methylsilyl ethers, a new approach to the problem was found. A protocol was developed for the direct gas chromatographic analysis of the amount of each anomer present in an aqueous solution. The protocol can be used on the micro scale and can be used in enzyme assays such as that for mutarotase. The method has been made more effective by combining gas chromatography with mass spectrometry. It is shown how mass spectral intensity ratios can be used to discriminate anomers one from another. The application of these methods to the study of complex mutarotations is discussed. 

Some 40 years ago Isbell (16) observed complex formation between calcium ions and a sugar which possesses the ax-eq-ax sequence—i.e., a-D-gulopyranose. He observed that an equilibrated solution of D-gulose, CaCl2, underwent further mutarotation on dilution with water and he correctly interpreted the phenomenon by postulating that a-D-gulo-pyranose (10), but not -D-gulopyranose, forms a complex with calcium... 

From the above discussion we infer that apart from the complexities of hydrolysis and mutarotation the most serious reservations concerning... 

With the establishment of the permease hypothesis, however, it was apparent that the mere formation of a complex with the mutarotase protein may be the necessary interaction in transport (15). The subsequent mutarotation could be considered to be a coincidental consequence of the complex formation. To support this idea, it was found that 1-deoxy glucose and a-methyl glucoside are excellent competitive inhibitors of the enzyme (16,61). Keston also showed that a number of cataractogenic sugars were inhibitors of lens mutarotase (62). It has since been shown that in all cases where a sugar is a substrate for the mammalian intestinal transport system it is also a competitive inhibitor of mutarotase. 

Addition compounds of sugars and sodium bisulfite are reported by Sundman.49 The rate of decomposition of these substances is primarily determined by the arrangement of hydroxyl groups. When the relationship is trans, more rapid decomposition is noted than when cis. The remainder of the molecule has little effect, although there is also a correlation between the rate of mutarotation and the value of the equilibrium constant. These observations are suggestive of a type of complexing similar to that of the borates. 

In the D-fructosides the convention is that the more dextro-rotatory anomer is the a form. Attempts have been made by Boeseken and Couvert,82 Verschuur83 and MacPherson and Percival84 to apply Boese-ken s boric acid method to determine the configuration of D-fructose at C2. The problem is much more complex than for aldoses because there are three hydroxyl groups near the reducing center, and also because the mutarotation of D-fructose involves the conversion of some pyranose to furanose form with the loss of a pair of cis hydroxyls (on C4-C5). This work has been discussed by Boeseken88 but no conclusion has been reached. 

It is of interest at this point to mention that the optical rotation of L-sorbose has been studied extensively. Lobry de Bruyn and Van Ekenstein21 reported that no mutarotation was observable however, Pigman and Isbell56 later discovered that L-sorbose does possess a small complex mutarotation. For example, at 20°C., L-sorbose (c, 11.3 l, 4) gave an initial observed rotation (°S) of —57.124, which changed to — 57.498 in 2.69 minutes and attained the final value of —57.768 after two hours. Similar results were observed at 0°C. but at a much slower rate. These authors were of the opinion that the smallness of this mutarotation was due to the fact that the equilibrium solution of L-sorbose is... 

The mutarotation of ribose was first observed by Phelps, Isbell and Pigman47 48 who studied both the d- and L-isomers in aqueous solution at 1°. The mutarotation of L-ribose in water at 0°, shown in Fig. 1, is a complex one, due probably to the existence of equilibria involving both furanose and pyranose forms. There is considerable evidence to support this view. Bredereck, Kothnig and Berger26 found that, while the mutarotation of D-ribose in pyridine at 20° is complex, the mutarotation of 5-trityl-D-ribose (which can exist only in the furanose form) in pyridine at 3° is of the normal, first order type. Isbell and Pigman48... 

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