Enzyme

Enzymes are biological macromolecules that act as specific catalysts during biological processes by making and breaking covalent bonds. The presence of active enzymes is essential to form most of the molecules in the cell. Many enzymes catalyse intracellular reactions which are too slow to be observed at all under comparable conditions in the absence of enzymes. Each enzyme makes one or more specific chemical reactions 10⁶ - 10¹² times faster, without affecting the final equilibrium concentrations of reactants and products. In addition, the enzymes are not broken down during the reactions they catalyse and they are very sensitive to changes in the chemical and physical environment

In thermodynamic language, enzymes reduce the activation energy of a reaction but do not affect the free energy change of the overall reaction. Enzymes are often highly specific, both for the molecules they will accept as substrates and for the precise chemical changes that they will catalyse. In general, an enzyme regulates the biochemical reaction pathway.

Enzyme reactions can be either anabolic or catabolic in nature^[1].

Enzymes and substrates must first interact to form an enzyme-substrate complex before any reaction can occur. This happens due to the motion and collision of molecules within cells though only a small proportion of collisions will result in a reaction. Enzymes will remain unchanged after catalysing the reaction.

Contents 1 The mechanism of Enzyme Action: 2 Specificity 3 Substrates and the Active Site 4 Allostery 5 Enzyme Types 6 Kinetics 7 Inhibition 8 Targets for drug action 9 References

The mechanism of Enzyme Action:

Enzymes increase the rate of reaction between 2 reactants in various possible ways:

• They improve the proximity of the substrate being that they increase the local concentration of the substrate. This increases the likelihood of substrate collision, resulting in an increases reaction rate.
• They affect the orientation and hold the atoms in positions that favour the reaction
• They produce strain distortion; they put strains on the bonds that are associated with the reaction
• In acid-base catalysis they aid in the exchange of H⁺ or generation of –OH^[2]
• They provide an alternative route of reaction with a lower activation energy so a higher proportion of substrate molecules collide with an energy above the activation energy and are able to move into the transition state.

2 models used to explain how enzymes work are stated as follows:

• the lock and key model - this is very simple and basically suggests that a substate with a shape complementary to an enzyme's active site will fit into it; and the reaction will take place to give products.
• Induced fit - this model suggests that as the active site of a given enzyme come into contact with its subtrate; it changes shape slightly to fit around the substate.

Specificity

Enzymes are very specific that they are able to distinguish between optical isomers.

The amino acids forming the active site mainly determine the specificity of the enzyme; a change in only a few amino acids in this region can result in a large change in the shape of the active site and this could then vastly change to affinity for the substrate or even change the substrate the enzyme is specific for.

The specificity of enzymes is exhibited in the ‘Lock and Key’ mechanism:

Image taken from http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=stryer&part=A1031^[3]

This illustrates the Lock and Key mechanisms and how the shape of the substrate is exactly complementary to the shape of the active site. The lock represents the enzyme and the key represents the substrate. Only a correct key (substrate) can bind to its corresponding lock (enzyme). However the lock and key model doesn't fully explain enzymatic activity. The model indicates that the enzyme and substrate are unable to change shape.

A modification to the Lock and Key Model of enzymes is the Induced Fit Hypothesis, also known as the "Hand-shake Model"^[4]. This hypothesis states that the structure of both the enzyme and substrate can change on binding. In essence, the enzyme can wrap itself around the substrate molecule, until the substrate is completely bound. This produces an enzyme-substrate complex, which places a strain on a particular bond, therefore weakening said bond; to a point where it can interact with the enzyme amino acid groups, further non-organic groups or further bound substrates.

The change in the shape of the enzyme is known as a conformational change; the purpose of which is two-fold:

1. As mentioned above, the conformational change places strain on the desired bond, allowing for a more efficient reaction to take place,
2. The new conformation brings amino acid groups essential to the enzyme reaction, which in the unbound conformation may distant from the active site, into the active site. These groups ensure the catalytic reaction will be optimal^[5]. The most common groups to be brought into the Active Site of the Enzyme are those relating to Acid/Base chemistry - therefore promoting the reaction and ensuring optimal conditions^[6].

Substrates and the Active Site

Whether an enzymatic reaction will occur is dependant on the substrate colliding and binding to the active site. Once a substrate binds to the active site, it is held there by a variety of interactions. These interactions take place between charged residual groups of the amino acids in the confirmed active site. Hydrogen Bonds and Ionic bonds generally occur - however they are very weak. These weak interactions are of the order of 3 - 12 kcal mol^-1 (12.5 - 50.2 kJ mol^-1) ^[7] this is of the order of 1/10^th the strength of an, on average, single covalent bond^[8].

This ensures that enzyme-substrate formation is a reversible process.

Allostery

An allosteric enzyme couples the effector levels to enzyme activity; it couples the signal to functionality. Allosteric enzymes have multiple binding sites (allosteric sites) and show cooperative binding^[9].

Allosteric control of enzymes can be positive or negative and can have effects such as up regulate or down regulate activity.

Types of Allosteric control:

1. Homotropic - The modulator is a substrate for the target enzyme as well as the regulator e.g. Oxygen acting on Haemoglobin.
2. Heterotropic - The modulator is the regulatory molecule but is not also the substrate of the enzyme.

Enzyme Types

There are many enzymes used in labs, each has it's own unique active site and so will catalyse a specific reaction. Restriction enzymes are one type of enzymes that are frequently used.

Restriction endonucleases are used naturally in a wide range of prokaryotes as a self-defence mechanism against foreign DNA molecules. The prokaryotes own DNA is methylated so it will not be cut by the enzyme.They recognise a specific 4-8 base pair palindromic sequence and by carrying out a hydrolysis reaction cut at that specific point. They may cut to form a blunt end or a sticky end. A blunt end is when the enzyme cut the DNA symmetrically. Asymmetrical cleavage leaves sticky end, these are unpaired bases. These sticky end can anneal to complementary bases on another strand^[10].

Kinetics

Kinetic parameters:

Two important enzyme parameters in a simple enzyme catalysed reaction are the Michaelis-Menten constant (K_m) and the maximum reaction velocity (V_max)

• K_m is the approximate measure of the enzyme affinity for the substrate. This can be calculated from the graph as ½ V_max. Generally, a lower K_m value signifies a higher affinity for the substrate.
• K_d is the dissociation constant for substrate binding to enzyme
• K_cat is the turnover number for the enzyme
• V_max is the maximal activity of the enzyme when all of the active sites are saturated.

The Michaelis-Menten equation:

V = V_max [S]/K_m [S]

To obtain V_max and K_m the enzyme activity must be recorded and then plotted on a double reciprocal plot, a Lineweaver-Burk plot, and the Michaelis Menten equation is then rearranged to look like this: 1/V = (K_m/V_max)(1/S)+1/V_max^[11].

Taken from www.search.com/reference/Lineweaver-Burk_plot^[12]A Lineweaver-Burk plot showing all the necessary parameters.

Inhibition

Enzymes can be inhibited by denaturing which is when a protein is changed in structure to form a randomly coiled peptide which exhibits none of its usual functions. Denaturing can result from extreme temperatures and pHs, as these alter the bonding in the molecule.

Inhibition can also be initiated by the binding of specific molecules called inhibitors. These can be split into categories:

1. Irreversible Inhibitors are molecules that permanently bind to the enzyme's active site or specific side chain, commonly to the serine (CH₂OH) or cysteine (CH₂SH) by covalent bonds. This inactivates the enzyme so the substrate cannot bind.
2. Competitive Inhibitors are competing for molecules that will have a very similar structure to that of the natural substrate and thus will be complementary to the enzyme active site. V_max stays the same, but K_m increases. This type of inhibition can be overcome by an increase in substrate concentration. They are therefore useful therapeutic agents and unlike irreversible inhibitors (like aspirin) their effect isn't long lasting.
3. Non-competitive inhibitors bind to the allosteric site on the enzyme other than the active site, causing changes to enzyme shape resulting in disruption of the active site. This decreases the turnover number of the enzyme rather than preventing substrate binding- V_{max decreases but Km} stays the same. This cannot be overcome with an increase in substrate concentration.
4. Uncompetitive inhibitors only bind to an enzyme-substrate complex, so both K_m and V_max decrease as it takes longer for the substrate to leave the active site. This inhibition works best when the concention of enzyme-substrate complex is high.We are able to distinguish the types of inhibition occurring by looking at the graph of enzyme activity^[13].

Image taken from http://ibhow.files.wordpress.com/2010/06/7-6-4.jpg?w=399&h=275 ^[14]

Competitive inhibitors show the same Vmax value however we see an increased K_m value and non-competitive inhibitors show a decreased V_max but the same K_m.

Targets for drug action

We are able to exploit these mechanisms of inhibition to engineer drugs with therapeutic effects.By targeting an enzyme in drug therapy we have the ability to change whole metabolic reactions that are catalysed by that particular enzyme. We can investigate possible new drugs by exploring drug-reaction interactions and drug-pathway interactions^[15]. Some examples of Drug Inhibitors:

• Irreversible drug inhibitors: Penicillin the antibiotic which inhibits transpeptidase in bacteria rendering them unable to synthesise cell walls. Aspirin which decreases the inflammatory response by inhibiting Cyclooxygenase.
• Competitive inhibitors: Methotrexate which inhibits dihydrofolate reductase which is involved in the synthesis of purines and pyrimidines.
• Non-competitive inhibitor: NNRT1 in the treatment of HIV^[16].

References

1. ↑ Nigel P. O. Green (1989). Biological Science. 2nd ed. Cambridge: Cambridge University Press. p.167.
2. ↑ Molecular Biology of the Cell 5th ed (2007) Alberts et.al. 159-169
3. ↑ http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=stryer
4. ↑ http://www.portlandpress.com/pp/books/online/glick/searchresdet.cfm?Term=induced-fit%20theory%20%28rack%20model%29
5. ↑ The World of the Cell, 3rd Edition, (1996) Becker et.al. p146, p147
6. ↑ The World of the Cell, 3rd Edition, (1996) Becker et.al. p146
7. ↑ Royal Society of Chemistry [RSC] http://www.rsc.org/ebooks/archive/free/SP9780851869209/SP9780851869209-FP015.pdf
8. ↑ The World of the Cell, 3rd Edition, (1996) Becker et.al. p146
9. ↑ J.Mol.Biol. (2004) 336, 263-273
10. ↑ Berg J., Tymoczko J and Stryer L. (2007) Biochemistry, 6th edition, New York: WH Freeman
11. ↑ Molecular Biology of the Cell 5th ed (2007) Alberts et.al. 159-169
12. ↑ http://www.search.com/reference/Lineweaver-Burk_plot
13. ↑ Biochemistry 6th (2006) Stryer et.al. pg51
14. ↑ http://ibhow.files.wordpress.com/2010/06/7-6-4.jpg
15. ↑ BMC Bioinformatics 2010, 11:501
16. ↑ Biochemistry 6th (2006) Stryer et al.