Mutarotase enzyme

Tphis review outlines the current knowledge of the enzyme mutarotase " (aldose-l-epimerase) and evaluates the evidence that it may have evolved from an origin in primitive bacteria into an important transport system for sugars in higher organisms. 

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. 

Bentley and Bhate140 conducted a meritorious study of the muta-rotations of D-glucose and D-galactose in the presence and absence of the enzyme mutarotase from Penicillium notatum under a variety of conditions. For measurements of the water-catalyzed and mutarotase-catalyzed reactions at 23-24°, they found the following... 

A mutarotase is an enzyme that oata lyses the irtter-conversfon of alpha and beta forms of... 

This enzyme [EC 5.1.3.3], also known as mutarotase, catalyzes the epimerization of the hemiacetal carbon atom of aldoses (thus, anomerization). Hence, a-D-glu-cose is reversibly converted to /3-D-glucose. Other sugars can act as substrates (e.g., L-arabinose, D-xylose, D-galac-tose, maltose, and lactose). 

The oxidation reactions of luminol and lucigenin can be used to assay for H Oj. For example, analysis of glucose in biological systems can be achieved using a three-enzyme system of mutarotase, glucose oxidase and horseradish peroxidase by correlation with the amount of HjOj released. Similarly, cholesterol can be measured using cholesterol oxidase. The fact that the rate of luminol oxidation depends on the concentration of the catalyst can be used as a method for determination of Co +, Fe +, Cr + and Mn + and other catalysts.Some examples of the use of luminol, isolumi-nol and their derivatives in immunoassays are shown in Table 3.11. ... 

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. 

Figure 5. Mutarotase assay by the GLC-TMSi method. The enzyme preparation was obtained from hog kidney, and the conditions are those described previously (26).

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. 

Bentley and Bhate (66) described the properties of the enzyme isolated from P. notatum, and Wallenfels and Herrman have reported the isolation and substrate specificity of a mutarotase from E. coli (63). The widespread occurrence of the enzyme in higher plants (53), fish (69), birds (70) and amphibia (69) has been noted. Sacks has recently described the properties of the enzyme in human red blood cells (75). 

Both approaches have been used for mutarotase and have given a considerable amount of information on the phylogenetic distribution of the enzyme and its relationship to developing sugar transport systems. 

Despite the broad spectrum of distribution, the catalytic properties of the enzyme in the different species are remarkably similar. The same four sugars (D-glucose, D-galactose, D-xylose, and L-arabinose) are substrates for the enzyme from all sources (Table VI). Some changes, however, have taken place, and the marginal activity for maltose found in the lower species (67) has been lost in the mammals, whereas the relative activity towards the pentoses has become enhanced. The Km values and turnover numbers for the substrates of purified beef kidney mutarotase are given in Table V. 

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). 

A detailed examination of the comparative kinetic properties of the enzyme from different species has been made (87). It appears that there are three kinetically distinct forms of mutarotase. The form with a Km of 12 mM is found in fish and some mammals, whereas the type with a... 

The relative amounts of the enzyme in kidneys of fish from different habitats have been measured (105) (Table IX). Particularly interesting is the finding that the mutarotase levels in kidneys of freshwater fish are always higher than those in saltwater fish, averaging six times greater. This extends an earlier observation by Keston that the enzyme was present in low levels in the aglomerular toadfish kidney (68). 

The anomeric preferences of these enzymes suggested that ratecontrolling anomerizations may occur during glucose metabolism, which could involve mutarotase. 

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