Structure of Enzymes | Biochemistry

In this article we will discuss about the structure of enzymes. This will also help you to draw the structure and diagram of enzymes.

Enzymes are proteins, having primary, secondary, tertiary and in certain cases, even quaternary structures. Some­times, this protein part (or apoenzyme) is not sufficient for catalytic action which then requires the presence of a co-factor.

I. Polypeptide Chains:

Some enzymes consist of a single polypeptide chain; in most cases these are secreted enzymes like ribonuclease. Other enzymes, in a much larger number, are composed of several chains (or sub-units), identical or different.

When enzymes comprise identical sub-units, each chain naturally carries an active centre: a tetrameric enzyme has 4 active centres. This category of oligomeric enzymes includes the allosteric enzymes (representing 10-20% of enzymes with quaternary structure).

In certain cases the dissociation into monomers is relatively easy; in others, stronger agents are needed to break the bonds linking the sub-units. It is only in very rare cases, that sub-units obtained by dissociation of an enzyme of quaternary structure, still possess activity.

Whether an oligomeric enzyme is allosteric (sigmoidal velocity curve) or not (Michaelian velocity curve), the interaction of sub-units within the oligomer is generally an absolute condition for the expression of catalytic activity.

The existence of isoenzymes i.e. enzymes existing in several molecular forms, should be mentioned: lactate dehydrogenase or lacticodehydrogenase for example, exists in 5 different forms which can be separated by electrophoresis.

These 5 isoenzymes are all tetramers which differ only by the mode of association of the two types of monomers (H and M); there are 5 possibilities of association: H₄, H₃M₁, H₂M₂, H₁M₃ and M₄. Different isoen­zymes of lacticodehydrogenase are found in the same organism depending on the tissue studied and changes in the distribution of these different forms are observed in certain pathological conditions.

Now, in a large number of cases, one knows not only the sequence of amino acids determined by the methods, but also details of the three-dimensional conformation thanks to the study of X-ray diffraction pat­terns of enzyme crystals.

One knows the three dimensional structure of hydrolases like lyzozyme, ribonuclease, chymotrypsine, and also that of dehydrogenases like alcohol dehydrogenase or glyceraldehyde-3-phosphate dehydrogenase, or that of kinases like hexokinase, or that of isomerases, etc.

The knowledge of the three-dimensional structures of these enzymes has led to significant advances in the study of the relationship between conformation and catalytic activity.

The molecular weight of enzymes is extremely variable, not only because of the presence of chains of varying lengths but also because of the presence of oligomeric enzymes consisting of a varying number of chains. It may vary from about 10 000 (ribonuclease: 13 700) to several hundreds of thousands (β-galac-tosidase: 520 000).

The enzymes which catalyze a series of metabolic reactions can aggregate and form multi-enzymatic complexes whose molecular weight can then widely exceed one million. For example, the transformation of pyruvate into acetyl- coenzyme A (see fig. 4-36) is made possible by a multi-enzyme complex com­posed of 3 different enzymes.

II. Co-Factors:

Some enzymes need a co-factor to exert their catalytic action.

There are different types of co-factors:

1. Metal Ions:

A large number of enzymes need a metal ion for activity; they are called metalloenzymes. The metal cation is supplied in the food to the cell which uses the metalloenzyme: it is an oligoelement. One of the ions most frequently found in metalloenzymes is Zn⁺: it is necessary for the expression of the activity of carbonic anhydrase, carboxypeptidase, alkaline phosphatases and a variety of other enzymes.

This metal is strongly bound to the enzyme (dissocia­tion constants of the order of 10^-10 M or less). In most cases, the Zn²⁺ ion participates in the recognition (K_m) and the catalytic transformation (V_m) of the substrate. Frequently, the binding of the cation Zn²⁺ to the enzymatic protein also plays a structural role by stabilizing an effective conformation of the active centre.

Some enzymes use Zn²⁺, but Mg²⁺, Ca²⁺ or even Na⁺ and K⁺ are also used by a very large number of other enzymes for activity.

Metalloen­zymes which contain iron or copper in their active centre are most frequently used for the catalysis of the oxidation-reduction processes:

2. Prosthetic Groups:

These are organic co-factors of non-protein nature strongly bound to the enzymatic protein, and whose presence is indispensable for catalytic activity. Thus, in catalases, peroxidases and cytochromes, the porphyrin group is covalently bound with the protein part (or apoenzyme), as in the case of heme and globin.

3. Coenzymes:

A. Mode of Action of Coenzymes:

These are also organic molecules, of small size compared to the enzymatic protein. These molecules are called coenzymes for historical reasons: they remained firmly bound to the enzymatic protein throughout the purification procedure.

This designation is extremely misleading. There are two broad categories of coenzymes. The first category which really deserves to be called coenzyme, includes the coenzymes which are really part of the active centre. In other words, the small coenzyme organic molecule assists the protein side chains in the catalysis. The best example of such a coenzyme is pyridoxal phosphate (fig. 7-1).

The second category of molecules called coenzymes does not really deserve its name. It would be preferable to call them substrates (or co-substrates). The prototype of this type of coenzyme is NAD⁺ (see fig. 2-16).

If a group X is transferred from a substrate “A” to a substrate “B” in presence of enzyme “E” and coenzyme “co”, there are 2 steps:

1. The Binding of X to the Coenzyme:

2. The Transfer of X to the Second Substrate:

The sum is written A – X + B A + B – X. It should be stressed that the coenzyme has reacted stoichiometrically and that it is regenerated.

There are two cases:

1. The above 2 steps are catalyzed by the same enzyme. In this case, the compound co-X formed intermediately is generally not released because the coenzyme remains bound to the enzyme. This takes place for example in reactions catalyzed by the transaminases whose coenzyme is pyridoxal phos­phate;

2. The 2 steps are catalyzed by 2 different enzymes. In this case the coenzyme is a transporter of X and it must be able to dissociate easily from its combina­tions with the two enzymes. These two enzymes therefore have a specific site for the reversible binding of the coenzyme which then behaves as a substrate.

This happens for example in the reactions catalyzed by the dehydrogenases whose coenzyme is NAD⁺ (see fig. 2-16).

B. Relations Between Vitamins and Coenzymes:

Vitamins (etymologically the term means: “nitrogenous substances needed for life”) are organic substances which cannot be synthesized by some or­ganisms and must therefore be supplied to them regularly but in small quan­tities — in order to ensure a normal metabolism. Certain vitamins, notably water-soluble vitamins and particularly the B group vitamins, are coenzymes or essential constituents of coenzymes.

It must be noted that the same compounds often serve as coenzymes for all living organisms, but are vitamins only for some of them. They may be compared with some amino acids which are found in proteins of all organisms but are “indispensable” or “essential” only for some of them (those which cannot synthesize them).

Even if a coen­zyme is not a vitamin for a given species, it can be one for an individual of this species having undergone a mutation affecting an enzyme needed for the synthesis of this coenzyme.

In animals and man, a prolonged deficiency in a vitamin of this type, called avitaminosis, produces a set of pathological symptoms due to the inability of the organism to catalyze some reactions (because the corresponding coenzyme is missing).

C. Principal Coenzymes:

Some coenzymes are of nucleotide nature. Coenzymes may be classified either according to their structure, or according to the reaction in which they participate (in the latter case distinction may be made between the coenzymes of oxidation-reduction reactions and those of group transfer reactions).

a) Coenzymes Involved in Oxidation-Reduction Reactions:

The following ones may be cited:

Nicotinamide Adenine Dinucleotide or NAD:

Its structure (see fig. 2-16) comprises 2 nucleotides (one consisting of adenine, ribose and phosphate and the other nicotinamide, ribose and phosphate) linked by a pyrophosphate bond.

Nicotinamide has the pyridine ring (this is why the coenzyme is also called Diphospho Pyridine Nucleotide or DPN) with a quater­nary ammonium; this explains why the coenzyme is written NAD⁺ when it is in oxidised form (in the reduced form nitrogen is ternary).

NAD is the coenzyme of various dehydrogenases; it binds the hydrogen originating from a substrate AH₂ and transforms into NADH + H ⁺ (sometimes written NADH₂ for brevity).

But in the course of another reaction catalyzed by a second dehydrogenase, it can transfer the hydrogen to another substrate B which will be reduced into BH₂, while the coenzyme is oxidized to the stage NAD⁺ (fig. 2-16). During these 2 reactions, the coenzyme really behaves like a substrate (just as A or B).

Just like the nucleotides constituting the nucleic acids, this coenzyme ab­sorbs in U.V. around 260 nm (in oxidized or reduced form), furthermore, the reduced form absorbs around 340 nm, so that the enzymatic reactions involving this coenzyme may be followed up by spectrophotometry.

Nicotinamide Adenine Dinucleotide Phosphate or NADP:

This is also a coenzyme of some dehydrogenases, very similar to the one mentioned above; it differs only by an additional phosphate residue esterified on the hydroxyl in 2′ of the ribose bound to adenine (it was also called TriphosphoPyridine Nucleotide or TPN). It is a hydrogen transporter. Its mechanism of action is very similar to that of NAD and therefore, it also behaves as a substrate.

The relationship of these 2 coenzymes with nicotinic acid or vitamin PP (“Pellagra Preventing” i.e. preventing a disease called pellagra) is rather evi­dent.

Flavine Nucleotides:

Flavine Mono Nucleotide (FMN) and Flavine Adenine Dinucleotide (FAD) are also coenzymes of dehydrogenases often called flavoproteins or yellow ferments (example: Warburg respiratory yellow ferment) because of their coloration due to the chromophore group formed by the conjugated double bond system of the flavine ring. As shown by figure 2-17, these two coenzymes derive from riboflavin or vitamin B₂.

It must be noted that FMN does not contain D-ribose; it contains a residue of D-ribitol (a penta-alcohol) and is not therefore, a true nucleotide. The figure also shows how they can participate in processes of cell oxidation by fixation – reversible — of 2H and formation of a colourless leuco-ferment (due to the disap­pearance of the conjugated double bond system. They behave like substrates. Succinate dehydrogenase, an enzyme of the Krebs cycle, is an example of FAD enzyme.

Ferro-Porphyrins:

These ferroporphyrin coenzymes are associated with cytochromes. In reduced form, iron is divalent; in oxidized form it is trivalent.

Lipoic Acid:

This coenzyme acts as hydrogen transporter in the oxida­tive decarboxylation reactions, particularly in that of pyruvic acid (structure and mechanism of action shown in figure 4-36).

b) Coenzymes Involved in Group Transfer Reactions:

One may cite: THIAMINE PYROPHOSPHATE (TPP) or CO-CARBOXYLASE – The pyrophosphoric ester of thiamine or vitamin B₁ is an integral part of the active site of carboxylases (hence its name). It is a transporter of aldehyde groups, particularly acetaldehyde. It is a coenzyme of decarboxylases of α-keto acids. Its structure and mode of action are described in connection with the decarboxylation of pyruvic acid.

Coenzyme A (Or Acylation Coenzyme):

As shown in figure 2-18, coenzyme A derives from a B group vitamin, the pantothenic acid, which is combined on the one hand, with a phosphate attached by a pyrophosphate bond to an adenosine 3′-5′ diphosphate molecule and on the other hand, to a thio-ethylamine (or cysteamine) molecule.

The thiol group of the latter is the active part of the molecule; its H atom can be substituted by various acyl radicals (especially acetyl) to form acyl-coenzyrnes A or activated fatty acids; chemically these are thio-esters, highly reactive compounds (acetyl-coenzyme A, for example).

We will study the importance of acyl-coenzymes A particular­ly in the oxidation of fatty acids (see figs, 5-10 and 5-11). We will also see that phosphopantetheine is the active part of a protein (ACP) transporting acyl radicals during the biosynthesis of fatty acids (see fig. 5-16). Coenzyme A behaves as a substrate.

Tetrahydrofolic Acid, FH₄:

It is the active form of a vitamin, folic acid or pteroylglutamic acid. In certain microorganisms, the synthesis of folic acid requires para-amino benzoic acid and that the bacteriostatic action of sulphamides is explained by a structural analogy of sulphamides with this compound, which enables them to bring about a competitive inhibition.

Tetrahydrofolic acid is a coenzyme, transporter of C₁ units (or one-carbon radicals), which behaves as a substrate and is involved in a large number of processes: biosynthesis of purine ribonucleotides (see fig. 6-19), formation of thymidylic deoxyribonucleotides, glycine ←→ serine intercon- version (see fig. 7-9) etc. Various derivatives of FH₄ can participate in these reactions; the structure of the main derivatives is given in figure 2.19.

S-Adenosyl Methionine:

It is a transporter of methyl groups (therefore, also of C₁ groups); it behaves as a substrate and in the presence of specific enzymes, it enables for example, the methylation of guanidino-acetic acid into creatine (see fig. 7-13) or that of certain nucleotides already incor­porated in the tRNA molecule (to form the methylated nucleotides called “rare” or “minor”). Figure 7-20 shows several methylation reactions involving S-adenosyl methionine.

Biotin:

It is the coenzyme of different carboxylation reactions. The carboxylation of acetyl-coenzyme A into malonyl-coenzyme A and we will see (see fig. 5-15) how biotin binds the CO₂ which it has to transfer and how it is linked by its carboxyl to ε-NH₂ of a lysyl residue of the enzymatic protein.

Pyridoxal Phosphate:

This coenzyme derives from pyridoxine or vitamin B₆. It is an integral part of the active site of various enzymes involved in the metabolism of amino acids: transaminases, amino acid decarboxylases, serine dehydratase, cysteine desulphydrase, etc. This shows that the same coenzyme can participate in completely different reactions depending on the en­zymatic protein to which it is associated.

Cobamide Coenzymes:

These coenzymes derive from cobalamine or vitamin B₁₂, whose structure is shown (see fig. 2-21) in the form of cyano- cobalamine (which is one of the most frequent), with a CN anion bound to cobalt. But other anions may be found in place of CN^– (CI^–, OH^–, NO₂^– etc.).

Besides, in place of the 5, 6-dimethylbenzimidazole molecule, linked on one hand to cobalt by a coordination bond and on the other hand, to a ribose-3- phosphate, one may sometimes find 5-hydroxybenzimidazole, or adenine. In coenzymes, in place of the anion, a 5′ deoxyadenosine is usually bound to cobalt.

The cobamides-coenzymes participate on the one hand, in various isomerization reactions, especially those implying a transfer of carboxyl group, as in the case of the isomerization of methylmalonyl-coenzyme A into succinyl-coenzyme A (see fig. 5-13), and on the other hand, in some methylation reactions.