• Enzymes are catalysts or chemical reagents that speed up chemical reactions without being consumed.
• Most enzymes are proteins that function to reduce energy of activation in chemical reactions.
• Because most enzymes are proteins, their activity is affected by factors that disrupt protein structure, as well as by factors that affect catalysts in general.
2. INTRODUCTION
Enzymes are catalysts or chemical reagents that speed up chemical reactions
without being consumed.
Most enzymes are proteins that function to reduce energy of activation in
chemical reactions.
They work on reactants called substrate; the enzyme attaches to the substrate
and then the enzyme converts the substrate into a product, while the enzyme
remains unaffected.
Because most enzymes are proteins, their activity is affected by factors that
disrupt protein structure, as well as by factors that affect catalysts in general.
6. The catalytic activities of enzymes are affected by multiple factors. They are,
TEMPERATURE
◦ The enzymatic activity will be optimum at normal temperature.
◦ At very low temperature the activity of enzyme will be minimal or zero.
◦ Increase in temperature can increase the enzyme’s catalytic activity.
◦ Maximum activity is observed is observed between 37 - 45˚C for most of the enzymes. This point
is known as optimum temperature.
◦ Beyond 45 ˚C the enzyme activity is reduced drastically.
◦ Beyond 60 - 70 ˚C the enzymes were denatured.
◦ The temperature co-efficient of majority of enzymes will be 2 at a temperature between 0 & 40 ˚C.
◦ Temperature co-efficient - Increase in the velocity of enzymatic reactions when the temperature is
increased by 10 ˚C.
7.
8. EFFECT OF LIGHT AND RADIATION
◦ Some enzymes are sensitive to light.
◦ Some enzymes require the presence of light for its catalytic activity.
◦ Example - Photolyase enzyme involved in the photoreaction DNA repair require light for its enzymatic
activity.
◦ Usually, enzymatic activity is reduced under the influence of harmful radiations such as X- rays, UV
rays, β rays and γ rays.
◦ Under these radiations, peroxides are formed which will cause oxidative stress.
9. WATER
◦ Enzyme activity is suppressed in the absence of water.
◦ In dry seeds, the enzymes are almost inactive.
◦ Hydration of the cells is necessary for the enzyme activity.
◦ Water provides a medium for the enzymatic reaction to take place.
◦ In many cases, water is one of the reactants.
◦ Hydration of lysozyme was determined by IR & NMR spectroscopy, first the charged polar groups of
the side chains hydrate, followed by the uncharged ones.
10. HYDROGEN ION CONCENTRATION (pH)
◦ Enzymes are protein substances that contain acidic carboxylic groups (COOH −) and basic amino
groups (NH +). So, the enzymes are affected by changing the pH value.
◦ Enzymes are active only over a limited range of pH
◦ Most of the enzymes are specific to a particular pH
◦ Each enzyme has a pH value that it works at with maximum efficiency called the optimal pH
Example:
Trypsin is active in alkaline medium.
Diastase is active in the neutral medium.
Pepsin show optimum activity in acidic pH
◦ Extreme pH values can cause enzymes to denature.
◦ Most enzymes work at neutral pH 7.4
11. ◦ Each enzyme will have an optimum pH where the velocity of the reaction is maximum.
◦ A bell - shaped curve is obtained for the effect of pH and enzymatic activity.
◦ Hydrogen ions in the medium influence the ionic charges on the amino acids in the enzymes
particularly on the active site.
◦ The hydrogen - ion concentration may alter the ionization and solubility of the enzymes, the
substrates, inhibitors and activators.
◦ It may also change the enzyme’s susceptibility to heat.
12. ACCUMULATION OF END PRODUCTS
◦ The end product accumulation retards the enzymatic activity.
◦ The active sites of the enzymes become crowded with the products.
◦ Thus, the substrate molecules will have comparatively lesser chances of combining with the active
sites.
◦ Inhibition by end products is also a regulation mechanism of the enzyme such as feedback inhibition
or allosteric modulation.
◦ Accumulation of products results in the increase in the rate of the reverse reaction.
◦ Inactivation of the enzyme because of the accumulation of the products at the reacting surfaces of the
enzyme itself.
◦ The pH of the products may be different from the initial pH and this may be unfavourable for the
enzymatic activity.
13. EFFECT OF ACTIVATORS
◦ Some enzymes require additional molecules for its optimum activity (generally known as prosthetic
groups or co - factors).
◦ Compounds which are active as prosthetic groups or which provide stabilization of the enzyme’s
conformation or of the enzyme - substrate complex.
◦ Rarely, anions are also needed for enzyme activity, (e.g.,) a chloride ion (Cl −) for amylase.
◦ The prosthetic groups may be inorganic metal ions such as Mg2+ , Cu2+ , Ca2+ , Na+ , K+ etc.. or
complex organic vitamin derivatives such as NAD, FAD etc..
◦ In the case of allosteric enzymes, there are molecules called allosteric modulators or effectors.
◦ Binding of these modulators to a specific site in the enzyme will cause more activated or less
activated forms of enzymes and thereby it can regulate the overall enzymatic reactions.
14. EFFECT OF INHIBITORS
◦ Inhibitors in the reaction can inhibit enzymatic activity.
◦ Type of inhibition depends on the nature of the inhibitors.
◦ Inhibitors are less effective when the concentration of enzyme and substrate is higher.
◦ Inhibitors are of different types:
Competitive Inhibitors - Competitive inhibitors compete with the substrate at the active site, and
therefore increase Km. However, Vmax is unchanged because, with enough substrate concentration,
the reaction can still complete.
• The graph plot of enzyme activity against substrate concentration would be shifted to the right due to
the increase of the Km, while the Lineweaver-Burke plot would be steeper when compared with no
inhibitor.
Non - competitive Inhibitors - Non-competitive inhibitors bind to another location on the enzyme and
as such decrease VMAX. However, KM is unchanged.
• This is demonstrated by a lower maximum on a graph plotting enzyme activity against substrate
concentration and a higher y-intercept on a Lineweaver-Burke plot when compared with no inhibitor.
15. Uncompetitive Inhibitors - Allosteric enzymes display a sigmoidal curve in contrast to the
hyperbolic curve displayed by Michaelis-Menten Enzymes. This is because most allosteric
enzymes contain multiple sub-units which can affect each other when the substrate binds to the
enzyme.
• Inhibition can affect either K0.5, which is the substrate concentration for half-saturation, Vmax or
both. This results in a shift of the curve to the right, and in the case of reducing Vmax, shifts the
curve down.
16. Comparison between Activator & Inhibitor
Enzyme Activator
◦ Chemical species can bind with an
enzyme to increase its activity.
◦ Effect on enzyme - Can increase enzyme
activity.
◦ Example - Include hexokinase - 1 &
glucokinase.
Enzyme Inhibitor
◦ Chemical species can bind with an
enzyme to decrease its activity.
◦ Effect on enzyme - Can decrease enzyme
activity.
◦ Example - Include drugs, ribonuclease
inhibitor.
17. Diagram to show the mechanism of both allosteric inhibition and activation
18. CONCENTRATION OF SUBSTRATE
◦ Increase in the substrate concentration increases the activity of enzymes until all the active
sites of the enzyme molecule are saturated with the substrate.
◦ After this saturation the rate of enzymatic reaction becomes steady.
◦ Then, the addition of the substrate will not have a positive effect on the velocity of reaction.
◦ Higher concentration of substrate can nullify the effects of competitive inhibitors in the medium.
Michaelis-Menten Kinetics
◦ Michaelis-Menten kinetics is a model of enzyme kinetics which explains how the rate of an
enzyme-catalysed reaction depends on the concentration of the enzyme and its substrate. 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
19. 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:
◦ This equation describes how the initial rate of reaction (V) is affected by the initial substrate
concentration [S]. It assumes that the reaction is in the steady state, where the ES concentration
remains constant.
20. ◦ This plot of rate of reaction against substrate concentration has the shape of a rectangular hyperbola.
However, a more useful representation of Michaelis - Menten kinetics is a graph called a Lineweaver -
Burk plot, which plots the inverse of the reaction rate (1/r) against the inverse of the substrate
concentration (1/[S]). The equation used to generate this plot is given by:
Graph of rate of reaction against substrate concentration, demonstrating Michaelis–Menten kinetics, with
Vmax and Km highlighted
21. CONCENTRATION OF ENZYMES
◦ Enzymes have extraordinary catalytic power.
◦ A small amount of enzyme is enough for large amount of substrate.
◦ Increase in the concentration of enzyme will increase the rate of reaction (if there is enough
amount of substrate is available in the medium).
◦ Increased number of enzyme molecules will have more active sites.
◦ Besides, at high concentration of the enzyme, the effect of inhibitors will be less.
22. CONCLUSION
◦ The activity of an enzyme can be measured by monitoring either the rate at which a substrate
disappears or at the rate at which a product forms.
◦ Enzyme activity is affected by factors such as temperature, pH, enzyme & substrate
concentration, water, activators & inhibitors, etc..
◦ These factors had significant influence on the rate of enzymatic activity.
◦ Every enzyme has different preferences of these factors. Therefore, every organism needs to
provide the appropriate conditions for all of its enzymes.
◦ Increase in substrate concentration leads to an increase in the rate of an enzyme - catalysed
reaction.
◦ Rate of an enzyme - catalysed reaction increases with an increase in the concentration of an
enzyme.
◦ An enzyme has an optimum pH range in which it exhibits maximum activity. Increase in
temperature increases the rate of an enzyme - catalysed reaction.
