Studies on Mutarotases: I. PURIFICATION AND PROPERTIES OF A MUTAROTASE FROM HIGHER PLANTS

Abstract

Abstract The enzyme mutarotase (aldose 1-epimerase), found in most mammalian tissues and certain bacteria, catalyzes interconversion of the anomeric forms of certain sugars, particularly those pentoses and hexoses related configurationally to d-glucose. This paper describes the widespread distribution of a similar enzyme in higher plants. The enzyme was found in both mono- and dicotyledons and was most widely distributed in the families Solanaceae (bell pepper, red pepper, tomato, eggplant), Cucurbitaceae (cantaloupe, cucumber, squash), and Liliaceae (asparagus, hyacinth, tulip). It was absent from all tested members of the Rutaceae (lime, lemon, orange, grapefruit) and Rosaceae (plum, pear, apple, strawberry). The mutarotase from green pepper (Capsicum frutescens) was isolated and purified approximately 1400-fold by a combination of salt fractionation, dialysis, preferential protein stabilization, and column chromatography on hydroxylapatite and diethylaminoethyl cellulose. The substrate specificity, in order of decreasing catalytic efficiency (d-glucose = 100), was d-galactose, 150; l-arabinose, 49; d-xylose, 18; and maltose, 6. The Km for d-glucose was 23.5 mm. Maltose, l-arabinose, and cellobiose were effective competitive inhibitors (Ki = 3.1, 6.3, and 12.3 mm, respectively). Phloridzin, phloretin, l-fucose, and l-deoxyglucose did not inhibit plant mutarotase. This is in marked contrast to the inhibitor specificity of the mammalian enzyme. The temperature optimum was 40°, and a broad pH-activity spectrum was displayed from pH 4.0 to 8.5, with the maximum at pH 7.4. The enzyme activity sedimented as a single band in sucrose density gradients and was eluted as a single peak from chromatographic columns. The molecular weight is approximately 50,000, and the turnover number is in excess of 8 x 105 moles of α- to β-glucose per min. The enzyme is sulfhydryl-dependent, being completely inactivated by 2 x 10-5 m mercuric chloride and reactivated by cysteine. A rapid, semiautomated assay procedure for the enzyme is also described.