Enzymes and Regulation of Protein Function

1. Enzymes
   
   • Features of enzymes
2. Active site
   
   • Lock and key model
   • Induced fit model
3. Enzyme kinetics
4. Graphs of enzyme kinetics
   
   • K_m
   • V_max
   • Lineweaver-Burk plots
5. Enzyme inhibitors
   
   • Irreversible inhibitors
   • Competitive, reversible inhibitors
   • Non-competitive, reversible inhibitors
6. Short-term regulation of proteins
   
   • Substrate and Product concentration
   • Allosteric regulation
   • Covalent modification
   • Proteolytic cleavage
7. Long-term regulation of proteins
8. Blood clotting cascade
   
   • Extrinsic pathway
   • Intrinsic pathway
   • Structure of prothrombin
   • Function of gla residues
   • Fibrinogen and formation of a fibrin clot
   • Regulation of the clotting cascade
   • Fibrinolysis
   • Important control points of the clotting cascade
9. Quiz

• Enzymes are biological catalysts that increase the rate of reaction by lowering the activation energy.
• Enzymes are highly specific molecules, most commonly proteins, that increase the rate of reaction whilst not being changed by the reaction and in some cases require cofactors.
• The active site of an enzyme is the site where the substrate binds and where the reaction takes place. There are two hypotheses as to how the enzymes fit their specific substrates: Lock & Key model and Induced Fit model.
• Enzyme kinetics can be shown on a graph which shows an enzyme catalysed reaction as it reaches a maximal velocity. By using the Michaelis-Menten model, the plot produces a rectangular hyperbola and you can work the maximal velocity (Vmax) plus the substrate concentration that gives half velocity (Km).
• Enzymes can be inhibited in multiple ways: competitively or non-competitively and reversibly or irreversibly. The action of the enzyme is affected in different ways depending on the inhibitor.
• Enzymes and other protein molecules can be regulated either in the short term or the long term.
• Short term regulation includes: substrate & product concentration, allosteric regulation, covalent modification and proteolytic cleavage.
• Long term regulation includes: altering the rate of protein synthesis and changing the rate of protein degradation.
• The blood clotting cascade is a highly regulated process and involves a number of types of regulatory mechanisms.

Enzymes

In chemical reactions, there is a minimum amount of energy that the substrates must have to allow the reaction to take place and lead to the generation of the products. This is called the activation energy.

Enzymes are biological catalysts that increase the rate of the reaction by lowering the activation energy required for the reaction to take place. Other ways of increasing the rate of the reaction are:

• Increased temperature - increases the number of molecules present with the activation energy.
• Increased concentration of substrates - increases the likelihood of molecular collisions.

Diagram - Graph of how enzymes reduce activation energy in a reaction

SimpleMed original by Dr. Peter Parkinson

Features of Enzymes

There are a number of features that enzymes have that enable them to carry out their function:

• Highly specific - enzymes have a specific shape that is complementary to a specific substrate and therefore only work on a specific reaction.
• Increase rate of reaction - by lowering the activation energy required for the reaction to take place, more molecules will be able to react together and the reaction can take place.
• Enzymes are usually proteins - most enzymes are sensitive to temperature changes and can become denatured at high temperatures as well as having reduced activity at lower temperatures
• Unchanged after the reaction - the structure of the enzyme is not changed by the reaction taking place therefore can be re-used in further reactions.
• Can require associated cofactors - cofactors can be a prosthetic group, which are permanently bound or a metal ion, vitamin or vitamin-made molecule (coenzyme), which are temporarily bound.
• Does not alter the reaction equilibrium - as the enzyme is not changed by the reaction, the reaction equilibrium between substrates and products is unchanged. The equilibrium is only altered by the thermodynamic properties of the substrates and products.

Active Site

The active site of an enzyme is where the substrate binds to and where the reaction takes place. There are some features of the active site:

• The active site only makes up a small part of the enzyme; most enzymes are more than 100 amino acids long, and the active site is formed by several amino acids that are brought together by the folding of the protein.
• Active sites are clefts or crevices where the substrate binds and water is excluded.
• The amino acids within the primary sequence of the polypeptide that form the active site are found at different points along the primary sequence.
• The bonds between the substrates and enzyme are weak, non-covalent bonds and the binding is not too tight. The bonds must be weak enough to allow the reaction to proceed and for products to be released. The weak bonds also allow the products to leave once the reaction is completed. 

There are two hypotheses as to how enzymes fit their specific substrate:

Lock and Key Model

• The active site of the enzyme is complementary in shape to the substrate and there is no further modification to the active site or substrate, just like how a key fits into a lock.

Diagram - The Lock and Key hypothesis

SimpleMed original by Dr. Peter Parkinson

Induced Fit Model

• The active site only becomes complementary in shape to the substrate once the substrate has bound to the active site.
• This model assumes that the active site is flexible and is similar to someone wearing a glove: the glove changes shape to fit the hand.
• Once the products have formed and moved away, the active site returns back to its original shape.

Diagram - The Induced Fit hypothesis

SimpleMed original by Dr. Peter Parkinson

Enzyme Kinetics

The Concentration of Substrate Decreases Over Time

• At the start of the reaction, the substrate concentration is at its highest. However, as the substrates are consumed in the reaction, the concentration of substrates decreases as the concentration of product increases.

Enzymes are Sensitive to Temperature

• The catalytic activity of enzymes is at its greatest at an optimal temperature of human body temperature (36.5°C-37.4°C).
• Above the optimal temperature, the bonds within the structure of the enzyme begin to break and the enzyme begins to denature therefore the rate of reaction begins to slow down.
• Some enzymes of different organisms have evolved to work at extremes of temperature. For example, the enzyme shown in blue on the graph could be from an organism living in the Arctic whereas the enzyme shown in red could be taken from an organism living in the hot springs of Yellowstone National Park in U.S.A.

Diagram - Graph showing the activity of different enzymes at different temperatures

SimpleMed original by Dr. Peter Parkinson

Different Enzymes Have Different Optimal pH

• A change in pH can change the ionisation state of the R groups of the amino acids present in the structure of the enzyme. This change in ionisation state can change the structure of enzyme and can lead to the enzyme becoming denatured.
• The optimal pH is not the same for each enzyme, as seen in the graph. The enzyme shown in green could represent pepsin which breaks down proteins in the acidic lumen of the stomach. Whereas, the enzyme shown in red could represent hexokinase present in the cytosol of human cells where it is a neutral pH. 

Diagram - Graph showing the activity of different enzymes with changes in pH

SimpleMed original by Dr. Peter Parkinson

Graphs of Enzyme Kinetics

We can display enzyme kinetic information using graphs by showing reaction rate as a function of substrate concentration. By doing this, the graphs show that an enzyme catalysed reaction reaches a maximum velocity.

The Michaelis-Menten model describes that a specific complex between an enzyme and the substrates is an essential intermediate step in the overall reaction.

The Michaelis-Menten equation predicts that a plot of velocity (V₀) versus concentration of substrate will produce a rectangular hyperbola, but it is important to remember not all enzymes will obey the Michaelis-Menten model.

Diagram - Graph showing reaction velocity versus substrate concentration with a rectangular hyperbola

SimpleMed original by Dr. Peter Parkinson

V_max = maximal rate when all enzyme active sites are saturated with substrate.

K_m = substrate concentration that gives half maximal velocity.

Importance of K_m Values

The value of K_m is often used as an indicator of the affinity of the enzyme for its substrate, although it also reflects aspects of the reaction kinetics.

• High K_m = Low affinity for the substrate
• Low K_m = High affinity for the substrate

If an enzyme has a low K_m value then less substrate is required to give half maximal velocity of the reaction due to the enzyme having a high affinity for the substrate.

Importance of Vmax Values

V_max and V₀ values are rates as they are measured in amounts per unit time.

1 unit = amount of enzyme that converts 1 μmol of substrate per min under standard conditions.

The value is expressed as a standardised rate - per litre of serum OR per gram of tissue.

It is important to remember that the rate of an enzyme catalysed reaction is proportional to the concentration of enzyme present therefore if you double the concentration of enzyme then the rate will double (NOT the standardised rate).

Lineweaver-Burk Plot

The Michaelis-Menten plot can be rearranged to change the rectangular hyperbola plot to a linear plot.

By changing the plot to a linear one, it allows for easier estimation of V_max and K_m values as well as easier comparison of two or more enzymes.

On the plot:

• The x-intercept equals -1/K_m. This means a more negative x-intercept indicates a low K_m and high affinity for the substrate.
• The y-intercept on the plot indicates 1/V_max. This means a low y-intercept value will indicate a high V_max value so fast maximal rate.
• The slope of the plot is calculated by K_m/V_max.

Diagram - A Lineweaver-Burk Plot

SimpleMed original by Dr. Peter Parkinson

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