Complex scientific questions concerning functioning of biological systems are usually approached as a series of subquestions that are easier to define and to investigate. When these subquestions have been solved, they provide insights, which illuminate the complexity of the entire organism. This approach has been successful so far and has led to the identification of the function of many proteins and other macromolecules. However, this approach does not allow elucidation of the complex interactions that occur in living cells and tissues and what the meaning of these interactions is for the functioning of the entire organism. The genome of man and other organisms has been sequenced, and we know in principle how many and which genes are involved, but this information does not give us a total functional map of all existing proteins. Additionally, alternative splicing of mRNA resulting in protein variations encoded by one gene and directed proteolysis for activation or degradation of proteins increases the multitude of functional proteins. The human genome comprises of approximately 30,000 genes encoding for, as is estimated now, 80,000 different proteins. Furthermore, many macromolecules in cells are not encoded for, such as lipids and sugars. These molecules play essential but often unknown roles in cellular processes as well. Finally, the intact biocomplex entity of a cell functions by virtue of a multitude of interacting molecular events. External stimuli or intracellular changes in gene expression, post-transcriptional and post-translational modifications modulate these interactions. On top of that it becomes more and more evident that proteins are multifunctional. These are all aspects that have to be taken into consideration as part of the complex regulation of the activity of proteins in general, and enzymes in particular.

Multifunctionality of proteins may be caused by the fusion of genes, thereby limiting the number of genes, but maintaining the different functions. Multifunctionality of proteins may be a common phenomenon. Molecular integration of metabolic and structural functions has many implications for cellular physiology such as cytoplasmic organization and cell motility and for the integration of structure and metabolism at the cellular level. Multifunctionality of proteins is called moonlighting, and adds another dimension to cellular complexity, from which cells can benefit in various ways. Therefore, elucidation of the functional roles of moonlighting proteins and their regulation is becoming a pertinent issue in cell biology as part of the functional proteomics approach. The function of a moonlighting protein can vary as a consequence of changes in cellular localization of a protein, cell type in which the protein is expressed, the oligomeric or polymeric state of a protein, and intracellular concentrations of ligand, substrate, cofactor or product of the protein. Moonlighting proteins can switch between functions in a number of ways as is demonstrated here with a few examples.

The Escherichia coli PutA protein has both proline dehydrogenase activity and pyrroline-5-carboxylate dehydrogenase activity when it is associated with the plasma membrane, but lacks enzymatic activity when it binds to DNA as a transcriptional repressor. Its localization switches on the basis of the amount of available substrate, ligand or cofactor. The PutA protein binds to the plasma membrane when substrate concentrations are high, but binds to DNA when substrate concentrations are low.

A protein can perform different functions when it is expressed in different cell types. Neuropilin is a receptor on endothelial cells for vascular endothelial growth factor. In axons, it serves as a receptor for another ligand, semaphorin III, which guides axons to find their destination during outgrowth.

Proteins can have different functions when they are present intracellularly or extracellularly. For instance, phosphoglucose isomerase is a ubiquitous cytosolic enzyme and catalyzes the second step of glycolysis. However, it is also secreted by cells and then it has at least four additional functions. Phosphoglucose isomerase can act as neuroleukin, which is both a cytokine that causes B cells to mature into antibody-secreting cells and a nerve growth factor that promotes survival of embryonic spinal neurons and sensory nerves. In addition, phosphoglucose isomerase/neuroleukin is also known as an autocrine motility factor, which is a cytokine that stimulates cell migration. Finally, the enzyme is a differentiation and maturation mediator that induces differentiation of human myeloid leukemia cells.

Some proteins have different functions when they are present as monomer or as multimer. For example, the monomer 37-kDa subunit of human glyceraldehyde-3-phosphate dehydrogenase acts as uracil-DNA glycolase in the nucleus, whereas the tetramer converts glyceraldehyde-3-phosphate into 1,3-diphosphoglycerate.

Complex interactions between enzymes and other macromolecules play important roles in regulating metabolic activity in vivo. Enzymes which were generally considered to be soluble and freely diffusable may be organized in multienzyme complexes or so-called molecular machines. It has become clear that well-defined intracellular compartments, such as cytosol and mitochondrial matrix, cannot simply be regarded as bags containing homogeneous solutions of enzymes, but are rather well organized. Furthermore, the role of water in metabolic processes is an interesting but largely ignored aspect in cell biology. Usually, enzyme reactions are measured in dilute solutions of cell or tissue extracts, whereas enzymes in vivo function in high (protein) concentrations in cells. Cells contain 20–40% proteins by weight. In this rich solution, interactions between enzymes and their cellular microenvironment are completely different from those in dilute solutions. Homologous and heterologous molecular interactions have been described to occur in vivo. Homologous interactions are considered to be changes in enzymes from a monomer form to a polymer form. Heterologous interactions are associations between enzymes and other proteins such as structural elements of the cell, e.g. membrane-associated structures or cytoskeletal components. These interactions may affect the catalytic activity considerably.

Studies by have shown that specific molecular interactions between aldolase, a glycolytic enzyme, and actin is responsible for the functioning of aldolase. Most of the aldolase is preferentially localized on stress fibers and in close vicinity of active ruffles of cells. This relative subcellular enrichment of aldolase is explained by its interactions with actin that can be modulated by physiological effectors such as insulin, calcium and anoxia. In the turtle brain, increased aldolase binding has been observed during anoxia-induced metabolic arrest. The physiological relevance of binding of aldolase to the cytoskeleton is still not clear, but it has been postulated that aldolase has a dual role as enzyme and as structural part of the cytoskeleton. Binding of aldolase to the F-actin core of microfilaments inhibits competitively the enzymatic conversion of the substrate of aldolase, fructose-1,6-biphosphate.

Post-translational regulation of metabolism by macromolecular interactions is distinctly involved in the activity of glucose-6-phosphate dehydrogenase (G6PDH). described the enzyme G6PDH in sea urchin eggs and found that in the unfertilized sea urchin eggs, 60% of G6PDH is bound to structural elements and then has a relatively lowV_max and high K_m whereas, within a few seconds after fertilization, G6PDH is largely released from its bound state and acquires a distinctly higher V_max and lower K_m. G6PDH is then able to produce large quantities of NADPH at the same physiological substrate and coenzyme concentrations that also existed before fertilization. NADPH is needed for hardening of the membrane (synthesis of the fertilization membrane) to prevent polyspermy and thus a lethal embryo, underlining the importance of rapid post-translational metabolic regulation in living cells. See example 10 for visualization of the immediate increase in NADPH production upon fertilization of sea urchin eggs.

Enzymes may switch partners and are then involved in different functions when their aggregation state or intracellular and/or extracellular localization is changed.