Physical characteristics and chemi-osmotic transformations of mutarotases from various species.

Abstract Previous studies indicated that the function of mutarotase (aldose-1-epimerase) in glucose metabolism in primitive organisms should have become redundant with the evolution of the phosphorylitic pathways of glucose utilization and suggested that in higher organisms the enzyme may have evolved into a component of a glucose transport system. In order to obtain some information on its evolutionary background, the enzyme was purified to homogeneity from a variety of species, including some mammalian embryonic forms, and the physical and catalytic properties were compared. Mutarotase content is highest in mammalian kidneys, comprising from 0.15 to 0.29% of the total soluble protein of this tissue. The catalytic coefficients (moles of α-glucose converted to β-glucose per mole of enzyme per min) of the purified enzymes ranged from 2.8 x 105 for rabbit to 1.0 x 106 for the bovine kidney enzyme, the most active mutarotase so far isolated. On the basis of Km values for glucose, mutarotases fall into two distinct groups: those with a Km of 19 mm (bovine adult, sheep, sheep embryo, hog, hog embryo, mouse, gerbil, and dog) and those with a Km of 12 mm (human, rabbit, toadfish, yellow perch, chicken, catfish, and newborn calf). In the ox, three different kinetic forms of the enzyme which have otherwise indistinguishable physical properties are present at different stages of development (embryo calf Km 5 mm, calf (0 to 6 months) 12 mm, and adult 19 mm). All forms of the enzyme have the same substrate specificity and are inhibited equally by inhibitors of glucose transport such as phloridzin, phloretin, and diethylstilbestrol. The molecular sizes and asymmetries of the enzymes were measured by sedimentation velocity in the ultracentrifuge, electrophoretic mobility, and gel permeation chromatography. All were monomers of the same size as indicated by the following values for the molecular weights: human kidney (37,500), bovine adult kidney (37,300), calf kidney (37,300), lamb kidney (38,100), embryo calf liver (38,000), rabbit kidney (36,700), chicken kidney (35,900), yellow perch kidney (35,800), guinea pig kidney (38,100), and green pepper (38,300). The Stokes radii averaged 31.4 ± 1.3 A, indicating similar molecular asymmetries. The corresponding frictional ratios (f:f0) in buffers of low osmolarity were about 1.37 and the enzymes from all species underwent an identical molecular transition, converting to a more compact form with an f:f0 of 1.17 in buffers of physiological ionic strength. This transformation was also monitored by measuring the changes induced in the filtration of purified bovine kidney mutarotase through Diaflo membranes and was shown to be specifically reversed by substrate sugars. The prolonged evolutionary history and apparently stable physical and catalytic properties of the enzyme, when taken in conjunction with the lack of a requirement in glucose metabolism, indicate that some significant alternative function must have evolved. The osmotic transformation undergone by all mutarotases may be relevant to this function, since it suggests a possible mechanism whereby sugar transport could be coupled to the osmotic gradients which are established during solute transport in kidney and intestine.