Mutarotation mutarotases

Many reactions that are promoted by enzymes can also be catalyzed by acids or bases or by both. An example is mutarotation, the reversible interconversion of the a- and (i-anomeric forms of sugars (Eqs. 4-1 and 9-86). This reaction is catalyzed by a specific mutarotase and also by inorganic acids and bases. 

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. 

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. 

Mutarotation of 0.3% solutions of the freshly dissolved sugars in 12 ml of 5 mM EDTA, pH 7.4 was followed. Significant differences in mutarotation rates (AK) in the presence and absence of 100 units of bovine kidney enzyme were expressed as the ratio AK/Ksp. Differences of less than 5% in these rate constants were not considered significant. Of the 18 sugars listed, nine have been tested previously as substrates for other mammalian mutarotases with essentially the same pattern as described here. The pattern of specificity indicates that a 3-point attachment of enzyme and substrate is necessary for catalysis of mutarotation. b Data from 72). 

Whatever the site of the enzyme may be, Keston et al. have recently produced fairly conclusive evidence that glucose, which is reabsorbed by the kidney, is exposed to mutarotase at some stage of the process (117). Glucose infused into the renal artery spills into urine when the renal threshold is exceeded in the same anomeric form as that administered, whereas reabsorbed glucose in the renal vein is mutarotated. Hill has also shown that the anomer infused in excess is excreted in excess (73). 

In aqueous solution, a mutarotational equilibrium of the two anomers is reached spontaneously - but not instantaneously - in which the ratio a/ 3 is 36 64 at a temperature of approximately 30 °C. In water, the rate is much lower than in buffer(ed medium) [354].The enzyme mutarotase accelerates mutarota-tion considerably. The rate at which the equilibrium is reached spontaneously depends greatly on pH, temperature and other solution components. 

Glucose oxidase is highly specific for -D-glucose. As noted earlier, 36% and 64% of glucose in solution are in the a- and P forms, respectively. Complete reaction of glucose therefore requires mutarotation of the a- to p-form. Some commercial preparations of glucose oxidase contain an enzyme— mutarotase—that accelerates this reaction. Otherwise, extended incubation time allows spontaneous conversion. 

With an excess of invertase and GOD in the enzyme membrane the total rate of sucrose determination is limited by the spontaneous mutarotation. Therefore the sensitivity towards sucrose is only about 10% of that for glucose (Scheller and Karsten, 1983). Kinetic (dl/dt) measurement even gives only 1% of the glucose signal at the same sucrose concentration. Application of coimmobilized mutarotase gives rise to an increase of the sensitivity by a factor of 6 for stationary measurement... 

Mutarotases (For a treatment of enzymological terms and concepts see Chapter 5). Mutarotases ensure that the mutarotation of sugars in vivo is much faster than spontaneous, and thus couples various transport systems and metabolic pathways. The relatively non-specific aldose mutarotases isolated... 

Keston305-307 showed that the kidney, liver, and lens tissues of many animals contain an enzyme that catalyzes the mutarotation of a- and /3-D-glucose and other sugars it was called mutarotase.3 8,309 This, or a similar, enzyme had earlier been found in preparations of D-glucose oxidase from Penicillium notatum by Bentley and Neu-berger310 and Keilin and Hartree.309 The enzyme was extensively purified by Bentley and Bhate.140 The enzyme is also widely distributed in plant tissues.311... 

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