Selective Oxidative Modification and Affinity Cleavage of Pigeon Liver Malic Enzyme by the Cu2+-Ascorbate System

Abstract

The role of metal ion in the enzymatic reaction entails providing a bridge between malate and the enzyme and functions as a second sphere complex with the substrate (Hsu et al., 1976). Therefore, identification of the ligands for metal binding is ultimately important in understanding the structure-function relationship of this enzyme. In most metalloproteins, the amino acid residues involved in metal binding are dispersed along the complete sequence (Regan, 1993). Without a three-dimensional crystal structure available, affinity cleavage at the putative metal-binding site by the metal-catalyzed oxidation system (MCO) 1 may be the optimal approach of reaching the above goal. Using this technique with the Fe 2ϩ -ascorbate system, Asp 258 is successfully identified in our previous study as one of the metal-binding sites (Wei et al., 1994), as confirmed by site-directed mutagenesis (Wei et al., 1995). In that study, some divalent metal ions were found to be capable of providing protection of the enzyme against Fe 2ϩ -induced inactivation. Among the divalent metal ions, only Cu 2ϩ was found to accelerate the Fe 2ϩ -ascorbate-induced enzyme inactivation rate. In this work, the inactivation of malic enzyme by Cu 2ϩ -ascorbate system is investigated. We demonstrate, for the first time, that at different pH values Cu 2ϩ -ascorbate system shows different specificities in protein modification and peptide bond cleavage. Taking the advantage of this selectivity, three more metal binding ligands of pigeon liver malic enzyme are successfully identified. Novel sequence motifs for the metal-binding site of malic enzyme are also deduced. EXPERIMENTAL PROCEDURESEnzyme Purification and Assay-Malic enzyme from pigeon liver was purified to apparent homogeneity according to published procedure (Chang and Chang, 1982). The enzyme activity was assayed by monitoring the formation of NADPH at 30°C as described previously (Chang et al., 1992).Enzyme Modification and Cleavage-The inactivation experiments were performed at 0°C by adding freshly prepared solutions of cupric nitrate (6 M) and ascorbate (20 mM) into the enzyme solution (0.97 M) in sodium acetate buffer (66.5 mM, pH 5.0). The progress of enzyme inactivation was monitored by assaying the enzyme activity in small aliquots withdrawn at the designated time intervals. For modification of the enzyme at different pH values, Bis-Tris (pH 4.0 -7.0) or Bis-Trispropane (pH 6.3-9.0) was used as buffer. Other experimental conditions are provided in the figure legends.For detecting the peptide bond cleavage, the samples withdrawn from the reaction mixture were added to EDTA solution (4 mM) to

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