Role of bivalent cations in the phosphoglucomutase system. 3. Structure-function relationships in the ternary enzyme-metal-substrate complex

E J Peck, W J Ray
1969 Journal of Biological Chemistry  
The formation of catalytically active enzyme.metal.glucose phosphate complexes involving the metal ion series, Zn 2 +, Co2+, Mg 2 +, Ni 2+ , Mn 2+ , Ca 2 +, and Cd 2 +, was studied by means of both ultraviolet difference and solvent perturbation spectroscopy. Metal-specific differences were observed in the three-dimensional structure of these complexes which suggest that the size of a crevice in the enzyme.substrate complex can be either reduced or expanded through binding of the appropriate
more » ... the appropriate bivalent metal ion. Furthermore, crevice size can be correlated with enzyme activity: the larger the alteration of crevice size on metal binding the lower the enzymatic activity, whether the change involves reduction or expansion in size. These observations suggest that the metal ion plays a dual role in the phosphoglucomutase system: it serves both as a catalytic entity and as a modifier of enzyme activity through its effect on enzyme structure. Phosphoglucomutase, EC, requires a bivalent metal ion for catalytic activity (1). Although a number of different metals satisfy this requirement, the maximum activity induced by the most efficient metal ion, Mg 2+ , is as much as 1000-fold greater than that induced by the least efficient metal tested, Zn 2+ (2). The first two papers (2, 3) of this series describe attempts to correlate differences in induced catalytic efficiency with various aspects of the metal-binding process, e.g. affinity of metal binding, structural changes accompanying the formation of the enzyme-metal complex, pH effects on the catalytic activity induced by the various metal ions. Although these studies provide interesting information with regard to the metal-binding process, they are singularly unsuccessful in terms of correlating catalytic activity with any property of the enzyme-metal complex observed thus far. The present investigation was undertaken to examine the formation of the ternary enzyme -metal glucose-phosphate complex produced by binding of metal ions to the enzyme. glucose-phos. phate complex. As in the previous work involving metal binding by the free enzyme, both ultraviolet difference spectroscopy and solvent perturbation techniques were used, and both metal-specific structural changes that accompany metal binding by the enzyme glucose-phosphate complex and metal-specific structural differences among the enzyme.metal glucose-phosphate complexes thus produced were investigated. EXPERIMENTAL PROCEDURE Materials-The phospho-form of phosphoglucomutase, E,' was prepared as described previously (4) and stored as a precipitate at 40 in a 60% ammonium sulfate solution, 0.1 M in sodium acetate buffer, pH 5. "Metal-free" phosphoglucomutase was produced by extensive dialysis against EDTA (instead of cyclohexyldiaminetetraacetic-acid) and Chelex-100 as described in the first paper of this series (2). A molecular weight of 62,000 (5) and an Em of 7.7 (6) were used in all calculations; based on these values, the molar extinction coefficient of phosphoglucomutase at 278 myz, e2s, is 47,700. Glucose-l-P, glucose-6-P, and glucose-1,6-di-P, were prepared as described previously (7, 8); note that throughout this report an equilibrium mixture of glucose-l-P and glucose-6-P (6) containing 1 mole % of glucose-1,6-di-P is referred to as glucose-P. All other reagents used were analytical grade or the purest grade available. The chlorides of all metals and of Tris buffer (Mann) were used except where otherwise indicated. Methods-Ultraviolet absorption difference spectroscopy and solvent perturbation spectroscopy were conducted in a Cary 15 double beam spectrophotometer with thermostated sample and reference cells. Spectral techniques were similar to those described in the second report of this series (3) and analogous to those described by Herskovits (9). The order of mixing of enzyme, substrate, and metals was not important in any of these studies (10). The abbreviations used are: Ep, the phospho form of phosphoglucomutase which possesses a phosphate moiety covalentlY bound to a seryl residue of the protein; Ep. glucose-P, the sum of all complexes with the stoichiometry enzyme. phosphate. glucose. in which the attachment of one phosphate group involves the enzyme and the other the glucose moiety; Ep-M-glucose-P, the equilibrium mixture of all complexes with the stoichiometl enzyme. phosphate2-glucose metal with the attachment of phosphate groups unspecified, viz. Ep. Zn-glucose-P is the sum of the equilibrium concentrations of Ep-Znglucose-l-P, ED -Zn glucose-1,6-di-P, and Ep.Zn-glucose-6-P. 3754 by guest on March 24, 2020 Downloaded from
pmid:5805396 fatcat:uaaqovbxrrb4dcev7cpkkm27ja