Tag Archives: multienzyme complexes

Multifunctional enzymes

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 the multienzyme complexes, a product of evolution to maximize the catalytic efficiency, providing advantages that you wouldn’t have if such enzymatic activities were present on distinct proteins dissolved in the cytosol.

CONTENTS

What advantages do multifunctional enzymes provide?

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.
Multifunctional enzymesAnd 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 glycolytic enzymes 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.

Examples of multifunctional enzymes

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

Acetyl-CoA carboxylase or ACC (EC 6.4.1.2), a biotin-dependent carboxylase, is composed of two enzymes, a biotin carboxylase (EC 6.3.4.14) 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 fatty acid synthesis, 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 2.3.1.85) 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.

Type I fatty acid synthase

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.

PRA-isomerase:IGP synthase

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 4.1.3.27). Anthranilate is phosphoribosylated to form N-(5’-phosphoribosyl)-anthranilate or PRA, in a reaction catalyzed by anthranilate phosphoribosyltransferase (EC 2.4.2.18); 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 5.3.1.24), PRA is isomerized to enol-1-o-carboxyphenylamino-1-deoxyribulose phosphate or CdRP. CdRP is converted to indole-3-glycerol phosphate or IGP, in a reaction catalyzed by indole-3-glycerol phosphate synthase or IGP synthase (EC 4.1.1.48). Finally, tryptophan synthase (EC 4.2.1.20) 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

Glutamine-PRPP amidotransferase or GPATase (EC 2.4.2.14) 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 6.3.4.16), 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.

CAD

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 2.1.3.2), and dihydroorotase (EC 3.5.2.3), 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.

References

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;303-8. 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):17857-71

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 [Google eBook]


Multienzyme complexes

Multienzyme complexes are discrete and stable structures composed of enzymes associated noncovalently that catalyze two or more sequential steps of a metabolic pathway.
They can be considered a step forward in the evolution of catalytic efficiency as they provide advantages that individual enzymes, even those that have achieved catalytic perfection, would not have alone.

CONTENTS

What advantages do multienzyme complexes provide?

During evolution, some enzymes evolved to reach a virtual catalytic perfection, namely, for such enzymes nearly every collision with their substrate results in catalysis. Examples are:

  • fumarase (EC 4.2.1.2), which catalyzes the seventh reaction of the citric acid cycle, the reversible hydration/dehydration of fumarate’s double bond to form malate;
  • acetylcholinesterase (EC 3.1.1.7), which catalyses the hydrolysis of acetylcholine, a neurotransmitter, to choline and acetic acid, which in turn dissociates to form an hydrogen ion and acetate;
  • superoxide dismutase (EC 1.15.1.1), which catalyzes the conversion, and then the inactivation, of the highly reactive superoxide radical, (O2.-), to hydrogen peroxide (H2O2) and water;
  • catalase (EC 1.11.1.6), which catalyzes the degradation of H2O2 to water and oxygen.

Then, the rate at which an enzymatic reaction proceeds is partly determined by the frequency with which enzymes and their substrates collide. Hence, a simple way to increase it is to increase the concentrations of enzymes and substrates. However, their concentrations cannot be high because of the enormous number of different reactions that occur within the cell. And in fact in the cells most reactants are present in micromolar concentrations (10-6 M), whereas most enzymes are present in much lower concentrations.
So, evolution has taken different routes to increase the reaction rate, one of which has been to optimize the spatial organization of enzymes with the formation of multienzyme complexes and multifunctional enzymes, that is, structures that allow minimizing the distance that the product of a reaction must travel to reach the active site that catalyzes the subsequent step in the sequence, being active sites close to each other. In other words, what happens is the substrate or metabolic channeling, that can also occur through intramolecular channels connecting the active sites, as in the case of, among the multienzyme complexes, tryptophan synthase complex (EC 4.2.1.20), whose tunnel was the first to be discovered, and bacterial carbamoyl phosphate synthase complex (EC 6.3.4.16).
Metabolic channeling can increase the reaction rate, but more generally, the catalytic efficiency, in several ways, briefly described below.

  • The diffusion of substrates and products in the bulk solvent is minimized, then their dilution and decrease of concentration, too. This leads to the production of high local concentrations, even when their intracellular concentration is low. In turn this leads to an increase in the frequency of enzyme-substrate collisions.
  • The time required by substrates to diffuse between successive active sites is minimized.
  • The probability of side reactions is minimized.
  • Chemically labile intermediates are protected from degradation by the solvent.

Another metabolic advantage of multienzyme complexes, similarly to what happens with multifunctional enzymes, is that they allow to control coordinately the catalytic activity of the enzymes that compose them. And taking into account that the enzyme that catalyzes the first reaction of a pathway is often the regulatory enzyme, it is possible to avoid:

  • the synthesis of unneeded intermediates, which would be produced if the sequence of reactions were regulated downstream of the first reaction;
  • the removal of metabolites from other pathways as well as a waste of energy.

Examples of multienzyme complexes

From what was said above, it is not surprising that, especially in eukaryotes, the multienzyme complexes, like multifunctional enzymes, are common and involved in different metabolic pathways, both anabolic and catabolic, whereas there are few enzymes that diffuse freely in solution. Below are some examples.

2-Ketoacid dehydrogenase family

A classic example of multienzyme complexes are the three complexes belonging to the 2-ketoacid dehydrogenase family, also called 2-oxoacid dehydrogenase family, namely:

  • the pyruvate dehydrogenase complex (PDC);
  • the branched-chain α-keto acid dehydrogenase complex (BCKDH);
  • the α-ketoglutarate dehydrogenase complex, also called 2-oxoglutarate dehydrogenase (OGDH).

These complexes are similar both from structural and functional points of view.
For example, PDC is composed of multiple copies of three different enzymes:

Then, PDC, both in prokaryotes and eukaryotes, has the basic E1-E2-E3 structure, a structure also found in the other two complexes. Moreover, within a given species:

And, although these enzymes are specific for their substrates, they use the same cofactors, namely, coenzyme A, NAD, thiamine pyrophosphate, FAD, and lipoamide.
In order to differentiate them, for the pyruvate dehydrogenase complex, the α-ketoglutarate dehydrogenase complex, and the branched-chain α-ketoacid dehydrogenase complex, they are indicated, respectively:

  • E1p, E1o and E1b (EC 1.2.4.4);
  • E3p, E3o, and E3b (EC 1.8.1.4).

Note: the eukaryotic PDC is the largest multienzyme complex known, larger than a ribosome, and can be visualized with the electron microscope.

The pyruvate dehydrogenase complex is the bridge between glycolysis and the citric acid cycle, and catalyzes the irreversible oxidative decarboxylation of pyruvate, an α-keto acid. During the reactions the carboxyl group of pyruvate is released as carbon dioxide (CO2) and the resulting acetyl group is transferred to coenzyme A to form acetyl-coenzyme A. Furthermore, two electrons are released and transferred to NAD+.

Multienzyme Complexes
Fig. 1 – The Five Reactions Catalyzed by the PDC

Even during the reactions catalyzed by the α-ketoglutarate dehydrogenase complex and the branched-chain α-keto acid dehydrogenase complex, respectively, the fourth reaction of the citric acid cycle, the oxidation of α-ketoglutarate to succinyl-CoA, and the oxidation of α-ketoacids deriving from the catabolism of the branched-chain amino acids valine, leucine and isoleucine, it occurs:

  • the release of the carboxyl group of the α-keto acid as CO2;
  • the transfer of the resulting acyl group to coenzyme A to form the acyl-CoA derivatives;
  • the reduction of NAD+ to NADH.

The remarkable similarity between protein structures, required cofactors and reaction mechanisms undoubtedly reflect a common evolutionary origin.

What are keto acids?

Keto acids or oxoacids are organic compounds containing two functional groups: a carboxyl acid group and a ketone group. Depending on the position of the ketone group, alpha-keto acids, beta-keto acids and gamma-keto acids can be identified.

  • Alpha-keto acids or 2-oxoacids have the ketone group at position α (2) from the carboxylic acid group, that is, adjacent to it. These compounds are important in biology, being involved in glycolysis, like pyruvic acid, the simplest α-keto acid, and in the citric acid cycle, like oxalacetic acid and α-ketoglutaric acid.
  • Beta-ketoacids or 3-oxoacids have the ketone group is at position β (3) from the carboxylic acid group. An example is acetoacetic acid, the simplest β-ketoacid, and one of the three ketone bodies, together with acetone and β-hydroxybutyric acid, produced by the hepatocyte in presence of an excess of acetyl-CoA, such as during fasting or low-carbohydrate diets.
  • Gamma-keto acids or 4-oxoacids have the ketone group is in position γ (4) from the carboxylic acid group. An example is levulinic acid, the simplest beta-keto acid, deriving from the catabolism of cellulose.
Keto Acids
Fig. 2 – Keto Acids

Tryptophan synthase complex

The tryptophan synthase complex is one of the best-studied examples of substrate channeling. Present in bacteria and plants, but not in animals, in bacteria it is composed of two α and two β subunits associated as αβ dimers, which are considered the functional unit of the complex, in turn associated to form an αββα tetramer.
The complex catalyzes the final two steps of the synthesis of tryptophan. In the first step, indole-3-glycerol phosphate undergoes an aldol cleavage, catalyzed by a lyase (EC 4.1.2.8) present on the α subunits, to yield indole and a molecule of glyceraldehyde 3-phosphate. Indole then reaches the active site of the β subunit via a about 30 Å long hydrophobic tunnel that, in each αβ dimers, connects the two active sites. In the second step, in the presence of pyridoxal 5-phosphate, a condensation between indole and a serine forms tryptophan.

Acetyl-CoA carboxylase

Acetyl-CoA carboxylase (ACC) (EC 6.4.1.2), a member of the biotin-dependent carboxylase family, catalyzes the committed step of de novo fatty acid synthesis, namely, the carboxylation of acetyl-CoA to malonyl-CoA, which, in turn, serves as a donor of two-carbon units for the elongation process leading to the synthesis of palmitic acid, catalyzed by fatty acid synthase (EC 2.3.1.85).
In bacteria, ACC is an multienzyme complex composed of two enzymes, biotin carboxylase (EC 6.3.4.14) and a carboxytransferase, plus a biotin carboxyl-carrier protein or BCCP.
Conversely, in mammals and birds, it is a multifunctional enzyme, as the two enzymatic activities, and BCCP, are present on the same polypeptide chain.
In higher plants both forms are present.

Carbamoyl phosphate synthetase complex

Another well-characterized example of substrate channeling is the bacterial carbamoyl phosphate synthetase complex, which catalyzes the synthesis of carbamoyl phosphate, needed for pyrimidine and arginine synthesis. The complex has a about 100 Å long tunnel that connects the three active sites.
The first active site catalyzes the release of the amide nitrogen of glutamine as ammonium ion, that enters the tunnel and reaches the second active site where, at the expense of ATP, is combined with bicarbonate to yield carbamate, that, in the last active site, is phosphorylated to carbamoyl phosphate.

References

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

Berg J.M., Tymoczko J.L., and Stryer L. Biochemistry. 5th Edition. W. H. Freeman and Company, 2002

Garrett R.H., Grisham C.M. Biochemistry. 4th Edition. Brooks/Cole, Cengage Learning, 2010

Hilario E.,  Caulkins B.G., Huang Y-M. M., You W., Chang C-E. A., Mueller L.J., Dunn M.F., and Fan L. Visualizing the tunnel in tryptophan synthase with crystallography: insights into a selective filter for accommodating indole and rejecting water. Biochim Biophys Acta 2016;1864(3):268-79. doi:10.1016/j.bbapap.2015.12.006

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):17857-71

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

Koolman J., Roehm K-H. Color atlas of Biochemistry. 2nd Edition. Thieme, 2005

Michal G., Schomburg D. Biochemical pathways. An atlas of biochemistry and molecular biology. 2nd Edition. John Wiley J. & Sons, Inc. 2012

Moran L.A., Horton H.R., Scrimgeour K.G., Perry M.D. Principles of Biochemistry. 5th Edition. Pearson, 2012

Nelson D.L., Cox M.M. Lehninger. Principles of biochemistry. 6th Edition. W.H. Freeman and Company, 2012

Perham R.N., Jones D.D., Chauhan H.J., Howard MJ. Substrate channeling in 2-oxo acid dehydrogenase multienzyme complexes. Biochem Soc Trans 2002;30(2):47-51. doi:10.1042/bst0300047

Rodwell V.W., Bender D.A., Botham K.M., Kennelly P.J., Weil P.A. Harper’s illustrated biochemistry. 30th Edition. McGraw-Hill Education, 2015

Voet D. and Voet J.D. Biochemistry. 4th Edition. John Wiley J. & Sons, Inc. 2011

Yon-Kahn J., Hervé G. Molecular and cellular enzymology. Springer, 2009

Welch G. R., Easterby J.S. Metabolic channeling versus free diffusion: transition-time analysis. Trends Biochem Sci 1994;19(5):193-7. doi:10.1016/0968-0004(94)90019-1

Zhou Z.H., McCarthy D.B., O’Connor C.M., Reed L.J., and J.K. Stoops. The remarkable structural and functional organization of the eukaryotic pyruvate dehydrogenase complexes. Proc Natl Acad Sci USA 2001;98(26):14802-07. doi:10.1073/pnas.011597698