Enzyme Basics

Enzymes are catalytic proteins

Proteins comprise up to 90% of the dry weight of a typical parenchyma cell. While some of these are structural, the proteins of interest here are catalytic. Catalytic proteins are called enzymes.

Enzymes share properties with all proteins

Of course catalytic proteins are in some ways no different from any others. They are synthesized from instruction in DNA. They have 1°, 2°, 3°, and maybe 4° levels of structure. They are heat labile. As macromolecules in general, to pass through membranes, they must be moved by vesicular transport (exocytosis, endocytosis, etc.). The exception to that, of course, is transport through the nuclear envelope by means of the nuclear pore complex we observed earlier.

Plant enzymes are produced by instructions in the DNA of the nucleus, chloroplast, or mitochondrion. The gene in the DNA is transcribed into an mRNA copy and this is translated on ribosomes to produce the protein. Generally nuclear mRNA is translated on cytoplasmic 80S ribosomes, and plastome and chondriome mRNAs are translated on stroma and matrix 70S ribosomes, respectively.

A closer view shows that this protein synthesis process involves three different macromolecules: DNA, RNA, and protein. Together they constitute the central dogma of genetics.

Let us recall that the sequence of nucleotides in the mRNA are handled one triplet at a time using the first two bases in the codon explicitly and the third base in at least some cases. Notice how the codons specify one of 20+ amino acids that are brought in to be attached to the end of the growing polypeptide chain.

The subunits of enzymes of course are amino acids, so here we examine the structure of amino acids. They are grouped here by the properties of their R-groups. The properties are critical to the structure of an enzyme.

These amino acids are linked together by means of a peptide bond which is actually catalyzed by a ribozyme (a catalytically-active RNA molecule) which is part of the ribosomal RNA-protein complex of both 70S and 80S ribosomes.

The sequence of amino acids (the primary structure) brings different functional R-groups of each amino acid together. These R-groups interact to cause a protein to coil, fold, pleat, and so on into a three-dimensional conformation.

Enzymes accelerate chemical reactions

What makes enzymes different from other types of protein is that they catalyze (accelerate) chemical reactions. The amount of acceleration can be as much as 8 to 12 orders of magnitude (uncatalyzed rate x 100,000,000). What is truly amazing is that in doing this acceleration, the enzyme also is very specific in both the molecules it can use as substrate, and the particular reaction that it accelerates in those substrates. Enzymes can distinguish stereoisomers and use only one of the pair as a substrate; they may accelerate a reaction involving only one seemingly insignificant atom on a huge substrate molecule. Even more important, enzymes function at temperatures, pressures, and pH ranges that are compatible with the entire scope of living organisms! Platinum is a great catalyst for breaking double-bonds...but it requires great heat in order to do so. It is in the catalytic converter in the exhaust stream of your car for this reason. There is no known life-form on earth that can operate in the temperatures required for platinum to work. Enzymes are amazing because they work inside living organisms!

Enzymes carry all of this out by means of their conformation. The protein structure of the enzyme includes an active site to which the substrate(s) can bind. The diagrams below show how the active site and a substrate combine. In this binding process, both enzyme and substrate are altered slightly in three dimensions. This alteration is called an induced fit. The example I will be using here happens to be polyphenol oxidase. It binds to the substrates: catechol (a polyphenol) and molecular oxygen. The union of these three molecules stresses their structures. As a result of this structural stress, the oxidation of catechol to ortho-quinone and the simultaneous reduction of oxygen to water is greatly accelerated.

For the rest of this discussion, I will be borrowing heavily from an article published some years ago about the structure of an enzyme called polyphenol oxidase. In laboratory you will be using this enzyme to study the specificity of this enzyme. Here is a reference and abstract of the article as well as the link to read it directly for yourself.

The enzyme observed here is polyphenol oxidase (PPO), but it is known by other names as well. The structure shown here was taken from Ipomoea batatas which you may know as sweet potato. After purifying and crystallizing this enzyme from the sweet potato, it was subjected to x-ray analysis and the images were subjected to software and sequence analysis that resulted in the following ribbon diagram.

Because a protein is typically a very long sequence of amino acids, the 1° structure of PPO is shown here as a long ribbon. This sequence spontaneously forms the α-helical and β-pleated sheet forms of 2° structure that you can observe here. The style of this ribbon diagram shows the helical sections boldly and the pleated sheet sections very subtly. The combinations of α-helix, β-pleated sheet are connected by turns and folds within the protein to give the enzyme a unique 3° structure as shown above. The secondary and tertiary structure of a protein, is of course due to the R-groups carried by the amino acids in the primary sequence. The hydrophobic R-groups fold to the interior of the protein, while hydrophilic R-groups fold to the exterior of the protein. The final conformation of the enzyme brings together R-groups that were distant in the primary sequence! The conformation is stabilized by disulfide bridges connecting distant sulfur-containg amino acids. Somewhere in this complex space is a small pocked known as the active site where the R-groups that are brought together can bind to a chemical, stress its bonds, and accelerate a chemical reaction.

The close-up of the active site of polyphenol oxidase shows that it uses six amino acids to chelate two copper ions. These are histidine which has a polar cyclic R-group with nitrogen atoms with unshared electrons to provide the chelating power not unlike how EDTA interacts with metal ions. One of these is distinguished because it is connected by a sulfide linkage to a cysteine in another part of the protein. The diagram also shows the oxygen atom of a water molecule the associates with the copper ions in the active site. Another feature to notice is how the copper ions in the active site are in a pocket with two sides having histidine chelators from two distant parts of the primary sequence.

In the diagram above, you find a stereo-pair showing the active site with a molecule of phenylthiourea (PTU) lodged in the active site. To make this, the x-ray imaging and software interpretation was achieved using PPO crystals that had been soaked in a solution of PTU. I will recommend looking mostly at the left half of the pair because it shows the PTU best. This is a molecule that binds into the active site and uses its sulfur atom to associate with the copper cofactors and its phenyl group to associate with an adjacent phenylalanine in PPO. Recall from chemistry that "like dissolves like"; in this case, the chemically-similar phenyl groups associate tightly. In the stereo pair in the figure above, the green areas show the movements in the conformation of the PPO when PTU is bound ("induced fit").

In the lower panels above, you can see an external 3-D diagram generated by software. The pink areas are the hydrophilic surface of the enzyme and the blue regions are areas that are hydrophobic. You can see that the active site receives the substrate in an area that is polar to the outside and non-polar to the inside. The PTU is shown in the active site, mimicking where the actual substrate for the catalyzed reaction binds. Because PTU binds the active site almost irreversibly, exposure of PPO to PTU renders the enzyme completely inactive.

The active site is usually a cleft in the conformation where a molecule called the substrate can bind. Interactions between the substrate and the active site are sometimes called "induced fit". Both the conformation of the enzyme and the shape of the substrate are altered slightly in this interaction. Textbooks often describe this as "lock and key," but as you well know, a key is not altered by a lock at any moment, so "induced fit" is a more accurate descriptor. The stresses applied to the substrate(s) in the active site ultimately reduce the energy required to cause a reaction and a particular product to be formed.

The x-ray imaging analysis has revealed that the active site chelates two copper ions that interact with the polyphenol substrate (catechol) and molecular oxygen in a series of steps. These steps result in removal of the hydrogen ions from a catechol molecule and binding them to one oxygen atom to make water. The diagrams above show how the oxygen and catechol bind at the active site, and how they interact with the copper cofactors in three steps resulting in the formation of ortho-quinone.

The article from which these diagrams were taken has provided huge insight into the mechanism of a reaction that has been known by humans for decades. Back in the 1970s (when I was an undergraduate) we were using PPO to study enzymes in biology courses and we even used PTU to block the reactions... but while we knew what went in and what was produced, we really did not know how the enzyme worked. These researchers figured out how to use modern technology and the binding of PTU to figure out how the enzyme likely binds the catechol and stresses it to accelerate its oxidation.

In laboratory, you will be using catechol and a range of structural analogs to see whether their structural similarities allow them to be alternative substrates, competitive inhibitors, or neither. We will not be using sweet potato (Ipomoea batatas), however. Instead we will be using white potatoes (Solanum tuberosum). Would the results be similar/comparable? It turns out that polyphenol oxidase (PPO) is a fairly ancient enzyme found in most organisms that have evolved on our planet!

Below is a sequence alignment of the two distant parts of the polypeptide chain that interact to form the active site to chelate the copper ions found in the enzyme from seven different organisms. You can see that the six histidines (green/cyan) are in fairly conserved positions. The cysteine that is involved in the active site is highlighted in yellow, and the phenylalanines are highlighted in pink. The obvious similarities in the active site among these various organisms, underscore that these features are required for this enzyme to function properly. Other amino acids in this enzyme may differ slightly or more significantly as "less than important" among the organisms given the gulf of time since their divergence from common ancestors in evolution.

The enzymes whose active sites are aligned above have different names. Polyphenol oxidase is perhaps the most descriptive name, as there are a range of substances that can bind and react as substrates and they are all polyphenols. They are all oxidized in the reaction. The authors of the study that provided the diagrams above, used the name catechol oxidase. However, by tradition, the enzyme in Neurospora (fungus) and humans is known as tyrosinase. The other species in the list are invertebrate animals that use this active site with copper ions for carrying molecular oxygen in their blood; the protein is known as hemocyanin. Below are some small pictures of the species whose protein active sites were compared in the alignments above.