Multifunctional Proteins (Molecular Biology)

Multifunctional proteins combine several autonomous functions on a single polypeptide chain. In this sense autonomy implies that each function is assigned to a different region or domain of the polypeptide chain. Multifunctional proteins contrast with multienzyme complexes, in which different polypeptides are noncovalently associated. The definition excludes enzymes that catalyze different reactions using the same active site, such as asparaginase functioning as a glutaminase, phosphoglyceromutase , which can catalyze three different reactions using the same reaction center, or cystathionine-g-synthase which can catalyze a whole series of analogous reactions because of its relative lack of substrate specificity. Allosteric enzymes are not considered multifunctional. Even though they possess binding sites for effector ligands (inhibitors or activators) situated in different regions of the polypeptide chain, they are defined only with respect to the catalytic function.

Multifunctional proteins generally have different catalytic functions residing in separate domains of the same polypeptide chain. A few examples in Escherichia coli are DNA polymerase I, phosphoribosylanthranilateisomerase-indoleglycerolphosphate synthetase ((1), (2)) (see TRP Operon), the aspartate kinases-homoserine dehydrogenases ((3), (4)), and chorismate mutase-prephenate dehydrogenase ( (5),(6)). In Neurospora crassa, the two functions of tryptophan synthase (see TRP Operon), namely the cleavage of indoleglycerol phosphate and the combination of indole with serine, reside on the same polypeptide chain, whereas in E. coli each is carried by an independent chain, noncovalently linked to the other. In this case, the E. coli enzyme is not a multifunctional protein but a multienzyme complex ( (7),(8)).

Other examples are the flavocytochrome b2 of Saccharomyces cerevisiae (9), a homotetramer that is both an l-lactate-cytochrome c-oxidoreductase (a flavoprotein) and a cytochrome b 2.

1. More than two Catalytic Functions may be Fused on the same Polypeptide Chain

DNA polymerase I from E. coli is a monomer endowed with three activities: DNA polymerase, 5′-3′ exonuclease, and 3′-5′ exonuclease (10). Carbamylphosphate synthetase from ascites hepatoma cells is a homotetramer also endowed with aspartate transcarbamoylase activity (in E. coli, these activities are carried by independent proteins) ((11),(12)). In N. crassa a single polypeptide chain, organized as a homodimer, is responsible for five of the biochemical reactions leading to the biosynthesis of the aromatic ring (13).

There are situations where the two systems coexist. The fatty acid synthetase of S. cerevisiae is a multienzyme complex ^b6) composed of two multifunctional proteins. One of the polypeptides carries the malonyl transacetylase, the b-hydroxyacyldehydrase, the enoyl reductase, and the palmityl deacylase activities, whereas the other polypeptide chain carries the b-ketoacylsynthase, the b-ketoacylreductase, and the acylcarrier protein. In combination with acetylCoA carboxylase, the complex catalyzes the condensation of acetyl subunits to the final product, palmitic acid (14). The avian fatty acid synthetase, which carries the same series of reactions, is a homodimer organized in a head-to-tail manner. Each of the identical polypeptides carry all of the above-mentioned enzymes (15).

2. Catalytic and Noncatalytic Functions may be Fused

In the case of cytochrome b 2 from calf liver microsomes, one domain of the protein represents the cytochrome b2 proper, whereas another domain is responsible for an anchor function (16). The homodimeric mammalian meromyosin has a domain endowed with ATPase activity, whereas another domain has a structural function (forming thick filaments) (17). Diphtheria toxin is a monomer whose amino terminal part ribosylates the mammalian elongation factor, whereas the C-terminal part is responsible for transporting the catalytic part across the membrane (18). The biotin repressor is a very interesting bifunctional protein of 321 amino acid residues that acts at two different levels. In addition to its repressor function, it is endowed with acetyl CoA carboxylase holoenzyme synthetase activity (19).

3. Noncatalytic Functions can be Fused

Human and bovine serum albumins consist of three homologous domains. They are endowed with independent binding and transport functions for tryptophan, bilirubin, and long-chain fatty acids (20). The Lac repressor, a homotetramer, has two independent domains, a small N-terminal domain that recognizes the corresponding operator DNA and a core that binds the inducer. The amino terminal part of the constitutive monomer mediates the assembly into tetramers (21). Other repressors also have separate domains for ligand binding and for specific DNA binding. Mammalian immunoglobulins have separate antigen binding and complement fixation sites (22).

4. Structural Evidence for a Multifunctional Protein

1. There should be more than one function on a single polypeptide chain.

2. Autonomy of these functions must be demonstrated by the existence of distinct domains on this polypeptide chain. Genetic analysis may produce evidence if it is possible to isolate mutants that are defective in only one function. Single-point mutations may result in the loss of more than one function. In this case, however, pleiotropic effects should be excluded. Conclusive evidence may be obtained by constructing a detailed genetic map, as done for several enzymes from E. coli ((23),(24) ).

3. A method providing convincing evidence is the isolation and characterization of fragments that have retained their individual function unimpaired. The N-terminal domain may be produced and isolated using nonsense mutants. Limited proteolysis is also useful because the hinge peptide that links individual domains is often very sensitive to proteolytic attack (25).

4. Chemical modification may affect one function without affecting another. For example, E. coli aspartate kinase I activity is destroyed by treating the enzyme with sulfhydryl reagents, whereas the other catalytic activity of the bifunctional protein, homoserine dehydrogenase, remains intact (26).

The proper physical methods are ultracentrifugation, gel filtration, and PAGE in with and without denaturing agents to determine the molecular weight of the native protein and the number of its subunits. Isoelectric focusing may provide indirect evidence that the subunits are identical. The number of autonomous functions must exceed the number of separable protein bands. The protein band must be homogeneous. Final proof is evidently provided by the total sequence of the protein or of the coding gene (cDNA in the case of eukaryotes).

Next post:

Previous post: