Mannosidase limpet, activation

S. Singh, M. Scigelova, and D. H. G. Crout, Glycosidase-catalysed synthesis of mannobioses by the reverse hydrolysis activity of alpha-mannosidase Partial purification of alpha-mannosidases from almond meal, limpets and Aspergillus niger, Tetrahedron Asymmetry, 11 (2000) 223-229. 

With a-D-mannosidase preparations in a high state of purity, the addition of 0.01% of bovine albumin to the assay mixture may lead to a small increase in activity (not more than 10%), probably by lessening denaturation of the enzyme. a-D-Mannosidase from marine-molluscan sources is, to a considerable extent, activated in the assay by chloride ion, and, to some extent, by certain other anions. Maximum activity is displayed by the enzyme from the limpet, P. vulgata, when 0.1 M sodium chloride is included in the incubation mixture46 (see Section 11,5 p. 412). Chloride ion has no effect on the activity of jack-bean or rat-epididymal a-D-mannosidase. 

Although a-D-mannosidase from mammalian, plant, and molluscan sources is dependent upon zinc for its catalytic activity, the addition of this ion has a marked effect in the enzyme assay only at those pH values where the active, protein-metal complex dissociates appreciably despite the presence of substrate. (Dissociation, which is greater at lower values of pH, is lessened in the presence of substrate.) The presence of zinc ion in the assay (0.1 mM) is thus of particular importance in the case of the limpet enzyme, where the pH of optimal activity is 3.5. Jack-bean and rat-epididymal a-D-mannosidase are both assayed at pH 5, and up to 10% activation may be observed with zinc. 

A somewhat different procedure was needed for purifying the limpet enzyme to the same extent.48 The activity of a-D-mannosidase from this source was rapidly destroyed by pyridine. Selective inactivation of 2-acetamido-2-deoxy-/3-D-glucosidase was, therefore, effected by warming the preparation in 60% ethanol. /3-D-Galacto-sidase was removed in the course of fractionation. 

With the exception of the enzyme from the limpet, P. vulgata, a-D-mannosidase from most of the important sources shows optimal activity at pH values lying between 4 and 5. For the enzyme from jack-bean meal39 and that from rat epididymis,80 we employed a pH of 5 for routine assays. If this is not the actual optimum, it is close to it on the broad pH-activity curves, and, at this pH, the addition of Zn2+ and other cations has relatively little effect in the assay, thus simplifying the study of the various metal complexes that can be formed by the enzyme protein. 

Fig. 1.—Hydrolysis at Different pH Values, in Acetate Buffer, of 6 inM p-Nitrophenyl a-D-Mannoside by Limpet a-D-Mannosidase.4S [A, no addition in the presence of A, 0.1 mM ZnS04 O, 0.1 M NaCl, and , 0.1 M NaCl + 0.1 mM ZnS04. The a-D-man-nosidase activity is expressed as a percentage of the maximum value obtained in the presence of Zn2+ and Cl-.]...

The firm binding of toxic cations by limpet a-D-mannosidase at pH 5 has already been mentioned (see Section 11,5 p. 412). This fact is not evident on assay at pH 3.5, because, at that pH, immediate exchange occurs with Zn2+ in the assay medium. On assay at pH 5 (used to arrest any cation exchange), an enzyme preparation may exhibit only about one-quarter of its potential activity at this pH. It is possible to accomplish replacement of endogenous, toxic cations by Zn2+, either directly by incubation with this cation, or indirectly, after incubation with EDTA (see Section III,4 p. 431). Subsequent assay at pH 5 then reveals activation. The lower the pH of incubation, the faster the removal of toxic cations.48... 

Activation of a-D-mannosidase from the limpet by Zn2+ and Cl-provides a particularly good example of the ways in which the kinetics of hydrolysis may be altered. Fig. 2 shows the effect of Zn2+, Cl-, or both, on the velocity of hydrolysis of substrate at varying concentration. Inspection of die curves reveals that Zn2+ increases the affinity of the enzyme for the substrate (competitive type of effect), whereas the main effect of Cl- is to increase the rate of hydrolysis (non-competitive effect). 

Apart from the effects of the strongly bound, toxic cations in the native preparation (see Section 11,5 p. 412), limpet a-D-mannosidase also resembles the jack-bean enzyme in its zinc-dependence. Fortunately, at the relatively low pH optimum (3.5) of the enzyme, the metals are freely dissociable, and toxic cations can be displaced by the addition of Zn2+. Full activity is only shown by the limpet enzyme at the pH optimum when an excess of Zn2+ is present in the assay medium (see Sections 11,3 (p. 408) and 11,5 (p. 412)]. 

This Section presents direct evidence for the conclusions arrived at indirectly from kinetic data (see Section 11,8 p. 416), namely, that Cl- accelerates the hydrolysis of the substrate by limpet a-D-mannosidase, whereas Zn2+ is an essential component of the enzyme in its catalytically active form. 

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