Introduction:
Enzymes are the organic biological catalysts, which speed up the rate of biochemical reactions without undergoing any change.
They are specific to one type of reaction and one, or a small number of, closely related reactants known as substrates.
Enzyme kinetics is the study of enzyme reaction rates and the conditions which affect them.
In this lecture, we will discuss the structure and function of enzymes, their clinical significance and theories of enzyme kinetics.
Structure of Enzyme:
Enzymes are proteins and usually have a globular tertiary structure.
Their structure is highly specific to the reaction they catalyze.
This is a small cleft within the enzyme with a specific amino acid structure allowing the substrate to bind and form the enzyme-substrate complex (ES), which is held together by weak bonds to allow dissociation of the complex when the reaction is finished.
The rest of the enzyme acts as a platform, bringing these key amino acids together.
The active site is always matching its specific substrate’s shape.
There are two main models for how this interaction occurs:
Lock and key model –
The active site is a perfect fit for the substrate and does not require any changes for it to bind.
Induced fit model –
The active site is almost complementary to the substrate but when it binds, the enzyme undergoes conformational changes to make its active site’s shape for a better fit.
This theory is more generally accepted than the lock and key model.
Enzymes have an optimal temperature and pH at which they work best, which varies depending on the enzyme's function as well as the enzyme's cellular and organ location.
Changes in pH can alter critical ionization states, whereas temperature changes can disrupt important bonds, affecting the structure and thus function of the enzyme.
If exposed to severe changes in temperature and/or pH, the shape of the active site may change.
This is referred to as enzyme denaturation and means the enzyme is useless for its biological function.
Enzyme Function:
Enzymes always choose a pathway for a reaction, which has a lower activation energy (Ea) – the minimum energy input needed for a reaction.
The transition state is a molecular intermediate between the substrate and its product, through which the reaction passes.
For example, in the equation below, X is the transition state. This transition state has a higher free energy than both the substrate and its product, however, the transition state is stabilized upon the addition of an enzyme.
Substrate → X → Product
Upon weak substrate binding, the enzyme’s active site changes shape such that it fits the transition state better than the initial substrate, hence has a higher affinity for this transition state.
This reduces the activation energy required to reach it.
Therefore, when the substrate binds to the active site, it is encouraged to continue the reaction and is converted into the transition state, and ultimately the final product of the reaction.
Rate-limiting Steps
The rate-limiting step of any reaction is its slowest step, and it determines the speed of the entire reaction.
In enzymatic reactions, the conversion of the enzyme-substrate complex to the product is normally rate-limiting.
The rate of this step (and therefore the entire enzymatic reaction) is directly proportional to the concentration of the enzyme-substrate complex.
The concentration of the ES complex changes as the reaction progresses, and therefore the rate of product formation also changes accordingly.
When the reaction reaches equilibrium (steady state phase), the ES concentration (and therefore the rate of reaction) remains relatively constant.
Reaction Kinetics
When an enzyme is added to a substrate, the reaction that follows occurs in three stages with distinct kinetics:
The pre-steady state phase is very short, as equilibrium is reached within microseconds.
In Michaelis-Menten Kinetics we actually measure the rate in a steady state.
Michaelis-Menten Kinetics.
Michaelis-Menten kinetics is an enzyme kinetics model that describes how the rate of an enzyme-catalyzed reaction is affected by the concentration of the enzyme and its substrate.
Let’s consider a reaction in which a substrate (S) binds reversibly to an enzyme (E) to form an enzyme-substrate complex (ES), which then reacts irreversibly to form a product (P) and release the enzyme again.
S + E ⇌ ES → P + E
To understand Michaelis-Menten kinetics the two terms we need to understand are:
Vmax:
The maximum rate of the reaction, when all the enzyme’s active sites are saturated with substrate.
Km (also known as the Michaelis constant):
The substrate concentration at which the reaction rate is 50% of the Vmax.
Km is a measure of the affinity an enzyme has for its substrate, as the lower the value of Km, the more efficient the enzyme is at carrying out its function at a lower substrate concentration.
The Michaelis-Menten equation for the reaction above is given as follows:
This equation describes how the initial substrate concentration ([S]) affects the initial rate of reaction (V).
It is assumed that the reaction is in a steady state with constant ES concentration.
When we plot a graph of substrate concentration versus reaction rate, we can see how the rate of reaction initially increases rapidly and linearly as substrate concentration increases (1st order kinetics).
The rate then stabilizes and increasing the substrate concentration has no effect on the reaction velocity because all enzyme active sites are already saturated (0 order kinetics).
Commonly Asked Questions.
What is the Michaelis-Menten equation? What is its application?
Write a short note on Enzyme Kinetics.
What is a rate limiting step? GIve one example.
Write a short note on the Structure of Enzymes.