Multifunctional enzymes are proteins in which two or more enzymatic activities, that catalyze consecutive steps of a metabolic pathway, are located on the same polypeptide chain. It seems likely they have arisen by gene fusion events, and represent, like multienzyme complexes, a strategy of evolution to maximize catalytic efficiency, providing advantages that you wouldn’t have if such enzymatic activities were present on distinct proteins dissolved in the cytosol.
- What advantages do multifunctional enzymes provide?
- Examples of multifunctional enzymes
Living organisms fight against the natural processes of decay that, if not counteracted, leads to an increasing disorder, until death. At the molecular level, the maintenance of life is made possible by the extraordinary effectiveness achieved by enzymes in accelerating chemical reactions and avoiding side reactions. The rate of ATP turnover in a mammalian cell gives us an idea of the rate at which cellular metabolism proceeds: every 1-2 minutes, the entire ATP pool is turned over, namely, hydrolyzed and restored by phosphorylation. This corresponds to the turnover of about 107 molecules of ATP per second, and, for the human body, to the turnover of about 1 gram of ATP every minute. Some enzymes have even achieved catalytic perfection, that is, they are so efficient that nearly every collision with their substrates results in catalysis.
And one of the factors limiting the rate of an enzymatic reaction is just the frequency with which substrates and enzymes collide. The simplest way to increase the frequency of collisions would be to increase the concentration of substrates and enzymes. However, due to the large number of different reactions that take place in the cell, this route is not feasible. In other words, there is a limit to the concentration that substrates and enzymes can reach, concentrations that are in the micromolar range for substrates, and even lower for enzymes. Exceptions are the enzymes of glycolysis in muscle cells and erythrocytes, present in concentrations of the order of 0.1 mM and even higher.
One of the routes taken by evolution to increase the rate at which enzymatic reactions proceed is to select molecular structures, such as multifunctional enzymes and multienzyme complexes, that allow, through the optimization of the spatial organization of the enzymes of a metabolic pathway, to minimize the distance that the product of reaction A must travel to reach the active site that catalyzes the reaction B in the sequence, and so on, thus obtaining the substrate or metabolic channeling of the pathway itself. For some multifunctional enzymes and multienzyme complexes the channeling is obtained through intramolecular channels.
Metabolic channeling increases the catalytic efficiency, and then the reaction rate, in various ways, briefly described below.
- It minimizes the diffusion of substrates in the bulk solvent, then their dilution; this allows to obtain high local concentrations, even when their concentration in the cell is low, thus increasing the frequency of enzyme-substrate collisions.
- It minimizes the time required by substrates to diffuse from one active site to the next.
- It minimizes the probability of side reactions.
- It minimizes the probability that labile intermediates are degraded.
Multifunctional enzymes offer advantages with regard to the regulation of their synthesis, too: being encoded by a single gene, it is possible to coordinate the synthesis of all the enzymatic activities.
Finally, like multienzyme complexes, multifunctional enzymes allow the coordinated control of their catalytic activities. And, because the enzyme that catalyzes the committed step of the sequence often catalyzes the first reaction, this prevents the synthesis of unneeded molecules, which would be produced if the control point were downstream of the first reaction, as well as a waste of energy and the removal of metabolites from other metabolic pathways.
Like multienzyme complexes, multifunctional enzymes, too, are very common and involved in many metabolic pathways, both anabolic and catabolic.
Here are some examples.
Acetyl-CoA carboxylase or ACC (EC 184.108.40.206), a biotin-dependent carboxylase, is composed of two enzymes, a biotin carboxylase (EC 220.127.116.11) and a carboxyltransferase, plus a biotin carboxyl-carrier protein or BCCP. ACC catalyzes the synthesis of malonyl-CoA by the carboxylation of acetyl-CoA. The reaction, which is the committed step of the synthesis of fatty acids, proceeds in two steps. In the first step, biotin carboxylase catalyzes, at the expense of ATP, the carboxylation of a nitrogen atom of biotin, that acts as a carbon dioxide (CO2) carrier, while the source of CO2 is bicarbonate ion. In the second step, carboxyltransferase catalyzes the transfer of the carboxyl group from carboxybiotin to acetyl-CoA to form malonyl-CoA. Malonyl-CoA is the donor of an activated two carbon unit to fatty acid synthase (EC 18.104.22.168) during fatty acid elongation.
In mammals and birds, acetyl-CoA carboxylase is a multifunctional enzyme, as biotin carboxylase activity and carboxyltransferase activity, plus BCCP, are located on the same polypeptide chain.
Conversely, in bacteria it is a multienzyme complex made up of three distinct polypeptide chains, namely, the two enzymes plus BCCP.
Both forms are present in higher plants.
Fatty acid synthase or FAS catalyzes the synthesis of palmitic acid using malonyl-CoA, the product of the reaction catalyzed by acetyl-CoA carboxylase, as a donor of two-carbon units.
There are two types of FAS.
In animals and fungi, it is a multifunctional enzyme, and is called type I. In animals it is an homodimer, and each polypeptide chain contains all seven enzymatic activities plus acyl carrier protein or ACP. In yeast and fungi FAS consists of two multifunctional subunits, called α and β, arranged in an α6β6 heterododecameric structure.
In most prokaryotes and in plants, fatty acid synthase, called type II, it is not a multifunctional enzyme but a multienzyme complex, being composed of distinct enzymes plus ACP.
The synthesis of the amino acid tryptophan from chorismate involves several steps, briefly described below.
In the first step, glutamine donates a nitrogen to the indole ring of chorismate, that is converted to anthranilate, and glutamine to glutamate; the reaction is catalyzed by anthranilate synthase (EC 22.214.171.124). Anthranilate is phosphoribosylated to form N-(5’-phosphoribosyl)-anthranilate or PRA, in a reaction catalyzed by anthranilate phosphoribosyltransferase (EC 126.96.36.199); in the reaction 5-phosphoribosyl-1-pyrophosphate or PRPP acts as a donor of a 5-phosphoribosyl group. In the next step, catalyzed by PRA isomerase (EC 188.8.131.52), PRA is isomerized to enol-1-o-carboxyphenylamino-1-deoxyribulose phosphate or CdRP. PRA e CdRP are an example of structural isomerism.
CdRP is converted to indole-3-glycerol phosphate or IGP, in a reaction catalyzed by indole-3-glycerol phosphate synthase or IGP synthase (EC 184.108.40.206). Finally, tryptophan synthase (EC 220.127.116.11) catalyzes the last two steps of the pathway: the conversion of IGP to indole, a hydrolysis, and the reaction of indole with a serine to form tryptophan.
In E. coli, PRA isomerase and IGP synthase are located on a single polypeptide chain, which is therefore a bifunctional enzyme. In other microorganisms, such as Bacillus subtilis, Salmonella typhimurium and Pseudomonas putida, the two catalytic activities located on distinct polypeptide chains.
Conversely, tryptophan synthase is a classic example of a multienzyme complex, and one of the best characterized examples of metabolic channeling.
Glutamine-PRPP amidotransferase or GPATase (EC 18.104.22.168) catalyzes the first of ten steps leading to de novo synthesis of purine nucleotides, namely, the formation of 5-phosphoribosylamine through the transfer of the glutamine amide nitrogen to PRPP. Note that glutamine acts as a nitrogen donor.
The reaction proceeds in two steps, which take place on different active sites, an N-terminal active site and a C-terminal active site. In the first step, the N-terminal active site catalyzes the hydrolysis of glutamine amide nitrogen to form glutamate and ammonia. In the second step, catalyzed by the C-terminal active site, which has phosphoribosyltransferase activity, the released ammonia is attached at the C-1 of PRPP to form 5-phosphoribosylamine. In this step the inversion of configuration about the C-1 position of the ribose, from α to β, occurs, then establishing the anomeric form of the future nucleotide.
There are three control points that cooperate in the regulation of de novo synthesis of purine nucleotides, and the reaction catalyzed by glutamine-PRPP amidotransferase, the first committed step of the pathway, is the first control point.
Like in bacterial carbamoyl phosphate synthetase complex (EC 22.214.171.124), the active sites of this multifunctional enzyme are connected through an intramolecular channel. However, this channel is shorter, being about 20 Å long, and lined by conserved nonpolar amino acids, then, it is highly hydrophobic. Lacking hydrogen-bonding groups, it does not impede the diffusion of the ammonia to the other active site.
The de novo synthesis of pyrimidine nucleotides occurs through a series of enzymatic reactions that, unlike de novo synthesis of purine nucleotides, begins with the formation of the pyrimidine ring, which is then bound to ribose 5-phosphate. The first three steps of the pathway are catalyzed sequentially by carbamoyl phosphate synthetase, aspartate transcarbamoylase (EC 126.96.36.199), and dihydroorotase (EC 188.8.131.52), and are common to all species.
In the first step, carbamoyl phosphate synthetase, which has two enzymatic activities, namely, a glutamine-dependent amidotransferase and a synthase, catalyzes the synthesis of carbamoyl phosphate from glutamine, bicarbonate ion and ATP. In the second step, which is the committed step of the metabolic pathway and is catalyzed by aspartate transcarbamoylase, carbamoyl phosphate reacts with aspartate to form N-carbamoyl aspartate. Finally, dihydroorotase, catalyzing the removal of H2O from N-carbamoyl aspartate, leads to the closure of the pyrimidine ring to form of L-dihydroorotate.
In eukaryotes, particularly in mammals, in Drosophila and Dictyostelium, a genus of amoebae, the three enzymatic activities are located on a single polypeptide chain, encoded by a gene derived from a gene fusion event occurred at least 100 million years ago. The multifunctional enzyme, known by the acronym CAD, is a homomultimer of three subunits or more.
Conversely, in prokaryotes, the three enzymes are distinct, and carbamoyl phosphate synthase is an example of a multienzyme complex.
In yeasts the dihydroorotase is present on a distinct protein.
Studies on enzyme activity have revealed the existence of a substrate channeling, more effective in yeast protein, with respect to the first two steps, than in that of mammals.
- Alberts B., Johnson A., Lewis J., Morgan D., Raff M., Roberts K., Walter P. Molecular Biology of the Cell. 6th Edition. Garland Science, Taylor & Francis Group, 2015
- Eriksen T.A., Kadziola A., Bentsen A-K., Harlow K.W. & Larsen S. Structural basis for the function of Bacillus subtilis phosphoribosyl-pyrophosphate synthetase. Nat Struct Biol 2000:7;303-308. doi:10.1038/74069
- Hyde C.C., Ahmed S.A., Padlan E.A., Miles E.W., and Davies D.R. Three-dimensional structure of the tryptophan synthase multienzyme complex from Salmonella typhimurium. J Biol Chem 1988:263(33);17857-17871. doi:10.1016/S0021-9258(19)77913-7
- Hyde C.C., Miles E.W. The tryptophan synthase multienzyme complex: exploring structure-function relationships with X-ray crystallography and mutagenesis. Nat Biotechnol 1990:8;27-32. doi:10.1038/nbt0190-27
- Michal G., Schomburg D. Biochemical pathways. An atlas of biochemistry and molecular biology. 2nd Edition. John Wiley J. & Sons, Inc. 2012
- Muchmore C.R.A., Krahn J.M, Smith J.L., Kim J.H., Zalkin H. Crystal structure of glutamine phosphoribosylpyrophosphate amidotransferase from Escherichia coli. Protein Sci 1998:7;39-51. doi:10.1002/pro.5560070104
- Nelson D.L., Cox M.M. Lehninger. Principles of biochemistry. 6th Edition. W.H. Freeman and Company, 2012
- Yon-Kahn J., Hervé G. Molecular and cellular enzymology. Springer, 2009