Enzyme

Enzymes act as specific catalysts. That is to say each enzyme accelerates one or more specific chemical reactions without affecting the final equilibrium concentrations of reactants and products. In thermodynamic language, enzymes reduce the activation energy of a reaction but do not affect the free energy change of the overall reaction. Many enzymes are so effective that they will catalyse intracellular reactions which are too slow to be observed at all under comparable conditions in the absence of enzyme catalysis. Enzymes are often highly specific, both for the molecules they will accept as substrates and for the precise chemical changes that they will catalyse, and the presence of active enzymes is essential to form most of the molecules in the cell.

Enzyme reactions can be either anabolic or catabolic in nature .  

Enzymes and substrates must first interact to form an enzyme-substrate complex before any reaction can occur. This happens through molecular motions where all of the molecules in a cell are constantly moving and colliding; however only a few collisions will result in a reaction.
The rate of encounter between the enzyme and the substrate is primarily dependant on the concentration on the substrate; meaning that, to increase the enzyme activity you must increase the substrate concentration.

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 yhey increase the local concentration of the substrate
• 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

Specificity

Enzymes are specific to the point of being 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:

Taken from http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=stryer&part=A1031

This illustrates the Lock and Key mechanisms and how the shape of the substrate is exactly complememtary to the shape of the active site.

Allostery

An allosteric enzyme couples the effector levels to enzyme activity; it couples signal to functionality. Allosteric enzymes have multiple binding sites (allosteric sites) and show cooperative binding.
Allosteric control of enzymes can be positive or negative and can have effects such as up regulate or down regulate activity.

Enzyme Types

There are many enzymes used in labs, one of which is the restriction enzyme.

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 .

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)
• Km is the approximate measure of the enzyme affinity for the substrate. This can be calculated from the graph as ½ Vmax. Generally a lower Km value signifies a higher affinity for the substrate.
• Kd is the dissociation constant for substrate binding to enzyme
• Kcat is the turnover number for the enzyme
•Vmax is the maximal activity of the enzyme when all of the active sites are saturated.

The michaelis-menten equation:

V = Vmax [S]/Km [S]

To obtain Vmax and Km 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 = (Km/Vmax)(1/S)+1/Vmax

Taken from www.search.com/reference/Lineweaver-Burk_plot

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 bind by covalent or non-covalent bonds to the enzyme.
2. Competitive Inhibitors are competing molecules that will have a very similar structure to that of the natural substrate and thus will be complementary to the enzyme active site. This type of inhibition can be overcome by an increase in substrate concentration.
3. Non-competitive inhibitors bind to a region on the enzyme other than the active site; this decreases the turnover number of the enzyme instead of preventing substrate binding. This cannot be overcome with an increase in substrate concentration.
4. Uncompetative inhibitors only bind to an enzyme-substrate complex; this also cannot be overcome by an increase in substrate concentration.
We are able to distinguish the types of inhibition occurring by looking at the graph of enzyme activity.

Taken from http://ibhow.files.wordpress.com/2010/06/7-6-4.jpg?w=399&h=275

Competative Inhibitors show the same Vmax value however we see an increased Km value and non-competitive inhibitors show a decreased Vmax but the same Km.

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. 

Some examples of Drug Inhibitors:

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

 

References