On the Importance of Being a Polymer

Part Two. Why are Enzymes Macromolecules?

23 JULY 2017,

The main point of this second part of the question “why macromolecules?” concerns the fact that the molecules directly responsible for our life, nucleic acids and proteins, are macromolecules – see examples in figure 1. The monomers, namely the building blocks, are the amino acids in the case of proteins, and the mono-nucleotides in the case of nucleic acids.

We have seen in a previous article that one general property of long polymeric chains is the tendency to form a condensed state, and in fact this is why, quite in general, synthetic polymers are important: because they tend to be “solid”. Thus, we can say something similar regarding our body: proteins of our muscles and skin and tendons, which are in fact called fibrous proteins, permit to give our body a certain consistency and robustness – also think of the wool, or silk, and hair, which are also proteins. And this is certainly a first kind of answer to the question “why macromolecules?” when thinking of proteins. However, the more sophisticated level is with the keyword enzymes.

Enzymes are proteins which are catalysts, namely they permit reactions to occur in the living cells, which would not proceed without them. Consider that in each liver cell, in this moment, thousands of reactions are going on, and each one is catalysed by a specific enzyme. The word “specific” is important: it means that a given enzyme is characteristic only for that particular reaction, and no other. So that in our life, animal and vegetal, thousands of enzymes are at work every moment, to guarantee the many processes of life.

And each of them is a macromolecule, a protein constituted by a long linear sequence of amino acids, as that shown in figure 1 (left side). Each linear sequence is generally folded in a particular spatial conformation, which is called native conformation, or native folding. Likewise, long sequence (of nucleotides, in this case) and a particular folding is displayed by nucleic acids also (see figure 1, right hand side). The question is: why long molecules? What there is so particular in the length, so that nature has been obliged to choose them to shape life?

Many good reasons to be a macromolecule

The most important reason for the macromolecular essence has been already given with the word “specificity”. Each enzyme has to recognize its own “substrate” without making errors.

And the binding takes place in a pocket, the so-called active site of the enzyme. It must be added that the binding of the substrate takes place without strong, covalent bonds – only weak secondary interactions are generally at work (the so-called hydrogen bonds, and electrostatic or hydrophobic interactions), and since they are “weak”, one needs many of them to generate a good binding force. But mostly, you can see from figure 2 that this interaction substrate/enzyme is in the form of a lock and key system, namely there is a three-dimensional tortuosity in the active site which is perfectly matched by the three-dimensional tortuosity of the substrate. And of course, to realize this special three-dimensional recognition, you need a lot of atoms, or, using a different language, many “bits of information”. This cannot be realized by, say, the interaction of the substrate with a simple linear dipeptide catalyst.

Now, notice from figure 2 (lower panel) that the amino acid residues involved in the active site are generally quite apart from each other in the primary structure – as indicated by the number attached to them. This is an important point: thanks to the dilution in a long, flexible chain, amino acid residues which are far apart in the primary sequence can come close to each other. This is the so-called “forced proximity”, as illustrated in figure 3.

Thus, this particular three-dimensionality constructs the magic of the active site, which permits the reactions to occur, usually with high speed at room temperature. The chemists do not always understand the details of the mechanism, but an important, general point, is the following: although the enzyme works – generally – in aqueous environment, the reaction within the active site does not necessarily take place in water. Actually, as shown in the simple-minded illustration of figure 4, the enzyme by binding the substrate, extracts it from water, and brings it in a very particular local environment, provided with a particular dielectric constant (usually lower than in water), which may facilitate reaction. This is the so-called “micro-environment” effect.

From here, let us go back to a point made previously on the basis of figure 2, about the fixed spatial conformation of the active site. How is the active site held in that particular spatial form? Actually, the whole enzyme has a “rigid” conformation, a term which indicates a profound difference between a functional biopolymer and a synthetic polymer in solution, say poly (vinyl chloride) or poly(ethylene). The synthetic polymers in solution acquire a “random-coiled” state, meaning by that the chains assume all possible conformations: if you would look at each single chain with a video-camera, you would see an extremely rapid change of the entire macromolecule from one spatial form to another one; and if you would take a static picture of the ensemble of the chains, (billions of them in a normal solution) you would not find two of them which have identical spatial form (see figure 5). Not so for a protein in solution: the enzyme conformation is held relatively rigid – the opposite of a random coil – thanks to a long series of intramolecular stabilizing interactions, as in the example shown in figure 5.

Actually, the non-obvious thing is, that only long chains (protein or RNA or DNA) can assume rigid conformations in solution – just because of the large number of possible intramolecular interactions – while short chains, like a tetra peptide or a tetra nucleotide, may not possess such a conformational rigidity. Only long chains can become “rigid”. Another good reason for being a macromolecule!

Now, let us look at a functional macromolecule like an enzyme or a RNA with a space-filling molecular model, as that shown in figure 6: we see in fact that the enzyme molecule is a spatial continuum, there are no hole or discontinuity in the structure. The X-ray protein specialists assert that proteins are tightly packed as good molecular crystals. And this has an important consequence: that every perturbation in any part of the molecule can be transmitted to all parts of the ensemble. This may induce conformational changes which in turn may facilitate the change into a different biological activity. This is the basis of the enzymes’ cooperativity, and/or allosteric behaviour, the most sophisticated mechanisms of enzyme regulation. We don’t have the time here to enter in the details of this quite fascinating subject. Instead, let us resume the following “four good reasons why it is a good for an enzyme to be a macromolecule”, by looking at the active site.
Binding – a long chain can give rise to a high stability of the enzyme-substrate complex via non-covalent weak interactions.
Stereochemical complementarity – only with a long chain can tortuous walls be built, which permit the good fit of the substrate.
Microenvironment – a long chain can create its own environment for the reaction.
Forced proximity of active residues – the active residues may be far apart in the primary sequence, which permits a high degree of freedom for the final adjustments required for catalysis.

Let us move now away from the active site on the surface of the enzyme. This is the contact region with the environment, water for aqueous soluble proteins, “oily” for membrane proteins, and obvious the structure of this periphery must be such, to maintain the fit with the environment.

The core of enzymes is generally “hydrophobic”, and generally, taken in isolation, would be insoluble in water. Therefore, this core must be surrounded by a hydrophilic layer, rich in negatively charged residues such as glutamate (with its –COO - group) and/or positively charged residues such as arginine or lysine (with their –NH3 + groups), to warrant solubility in water: additional “bits of information” are needed, which brings to a further enlargement of the macromolecule.

One could mention additional reasons, such as the multiple binding of more than one substrate (e.g. coenzymes), and the need of physical properties such as viscosity and hydrodynamic properties, but the general message is by now clear: an enzyme must perform many functions, has many chemical and physical properties, each of these necessitates a certain piece of structure, and many properties sum up in a long chain.

This question “why macromolecules” is part of a broader question in biology, which is: why in nature things are the way they are? Could have not been different? Usually, the analysis of this question ends up with the conclusion that yes, nature was right to do the way she did it. But in making this analysis, there is a lot that we are going to learn. More details on this argument (and related questions still open) can be found in my recent book, The Emergence of Life (2016).

References P. L. Luisi, [On the Importance of Being a Polymer. Part One: The Synthetic World]. Wall Street International – Science & Technology, 23 June 2017.
P. L. Luisi, The Emergence of Life. From Chemical Origins to Synthetic Biology, 2nd Edn., Cambridge Univ. Press, 2016.

Read also the First Part.