Malic enzyme of pigeon liver is a tetrameric molecule with identical, or nearly-identical subunits. It catalyzes, in addition to oxidative decarboxylation of L-malate, the following metal activated component reactions: Oxalacetate decarboxylase; reductase with broad specificity on α-ketocarboxylic acids; a NADP+-dependent dismutation of L-malate to L-lactate; and proton exchange between pyruvate and medium water. The kinetic mechanism of oxidative decarboxylase is sequential and ordered, with NADP+ adding first to the metal enzyme, followed by L-malate, and by the release of products CO2, pyruvate, and NADPH. NADPH release, or a conformation change preceeding it, is rate-limiting in the overall reaction. Chemical modification studies indicate the presence of histidyl and lysyl residues at the nucleotide site, and tyrosyl residues at the carboxylic acid site. The involvement of protonated histidine(s) in NADPH binding is implicated by results of direct titration experiments, which also suggest a role of this residue as a proton sink in the catalytic reaction. A cysteinyl SH group is located near (but not at) each of the substrate-sites on the enzyme tetramer. Reaction of these groups with SH reagents causes selective loss of activities involving decarboxylation (i.e., oxidative decarboxylase, reductive carboxylase, and oxalacetate decarboxylase), owing to blockage of the reversible carbon-carbon cleavage step by the bulky substituent. All-of-the-sites reactivity is observed for non-specific thiol reagents such as 5,5′ dithiobis-(2-nitrobenzoic acid), N-ethylmaleimide, iodoacetate, and iodoacetamide. While bromopyruvate, which is reduced by the enzyme to L-bromolactate under catalytic conditions, alkylates these groups in an active-site directed manner with half-of-the-sites stoichiometry. The remaining two SH groups are reactive toward non-specific reagents, but at rates 2.4 - 3.6 fold lower than do the same groups on the unalkylated enzyme. This behavior is interpreted in terms of the ligand-induced negative cooperativity concept of Koshland, et al. (Biochemistry 5: 365–385, 1966): Reaction of bromopyruvate induces a conformation change on the alkylated subunit which is transmitted to the unoccupied subunit neighbor, turning off its catalytic site for reaction with L-malate, as well as converting the initial ‘fast’ SH groups into ‘slow’, or unreactive SH groups. In equilibrium binding experiments, all-of-the-sites reactivity is seen with nucleotide cofactors NADP+ and NADPH. Binding of Mn2+, or L-malate in the presence of Mn2+ and NADPH is biphasic, showing two ‘tight’ sites with dissociation constants in the micromolar range, and two ‘weak’ sites with 10–100 fold lower affinities. The presence of ‘tight’ and ‘weak’ L-malate sites is confirmed by fluorescence titration experiments which also yields similar affinities for the substrate molecule. In kinetic studies, two types of non-equivalent, and functionally distinct sites are detected. At saturating NADP+, and Mn2+ and L-malate levels corresponding to binding at tight sites, typical Michaelian behavior is observed. The reaction is inhibited uncompentitively by L-malate at higher concentrations corresponding to occupancy at all of the L-malate sites. Occupancy of Mn2+ at weak metal sites as well has no effect at low L-malate, but prevents substrate inhibition at high L-malate. A tentative ‘half-of-the-sites’ model consistent with results of chemical modification, binding, and kinetic experiments is proposed for this enzyme. This model implicates involvement of subunit cooperativity in the catalytic process. Malic enzyme is depicted as a tetramer composed of inititally identical subunits, each containing an active-site capable of binding all reactants. Mn2+ and L-malate bind anticooperatively to the tight and weak sites, in contrast to NADP+ which binds equivalently to all sites. On the fully active enzymes, only half (or the tight) of the subunits are simultaneously undergoing catalysis. Binding of L-malate (but not Mn2+) at the adjacent weak subunits causes a slow isomerization of the enzyme, and inhibition of NADPH dissociation from the catalytic subunits. Binding of Mn2+ at the same sites prevents this change and thereby relieving substrate inhibition. This model is further supported by results of active-site titration experiments, such as the half-size burst of enzyme-bound NADPH in the transient state, and half-of-the-sites reactivity of oxalate, an analog for the transition state intermediate of the reaction.