Module 2. Enzymes

Lesson 10


10.1 Introduction

An enzyme assay measures the conversion of substrate to product, under conditions of cofactors, pH and temperature at which enzyme is optimally active. An enzyme is most conveniently assayed by measuring the rate of appearance of product or the rate of disappearance of substrate. If the substrate absorbs light at a specific wavelength, then changes in concentration of these molecules can be measured by following the change of absorbance at this wavelength. Typically this is carried out using spectrophotometer since absorbance is proportional to the rate of enzyme activity in moles of substrate used (or product formed) per unit time.

10.2 Enzyme Velocity

The rate of an enzyme catalyzed reaction is often called its velocity. It is normally reported as values at time zero ( V0 ; micomoles /min) since the rate is fastest at the point where no product is yet present. This is because the substrate concentration is greatest before any substrate has been transformed to product. A typical plot of product formed against time for an enzyme catalyzed reaction show an initial period of rapid product formation which gives the linear portion of the plot. This is followed by a slowing down of the enzyme rate as substrate is used up during the reaction.


Fig. 10.1 Rate of enzyme-catalyzed reaction with time

10.3 Enzyme Unit

Enzyme activity may be expressed as μmol of substrate transformed per minute ( μmol min-1). The standard unit of enzyme activity are enzyme unit and katal (kat). An enzyme unit is that amount of enzyme which catalyse the transformation of 1 μmol of substrate per minute at 25oC under optimal conditions for that enzyme. The katal is the SI unit of enzyme activity and is defined as that catalytic activity which will raise the rate of reaction by one mole per second in a specified system,. 1U=16.67 nanokatal. The term activity refers to total units of enzyme in the sample, whereas the specific activity is the number of enzyme units per milligram of protein (Units/mg).

10.4 Substrate Concentration

The normal pattern of dependence of enzyme rate on substrate concentration (S) is that at low substrate concentrations a doubling of (S) will lead to doubling of intial velocity (V0). However, at higher substrate concentration the enzyme becomes saturated, and further increase in (S) leads to very small changes in V0 this is called Vmax . This occurs because at saturating substrate concentrations effectively all of the enzyme molecules have bound to substrate. The over all rate is now dependent on the rate at which the product can dissociate from the enzyme, and adding further substrate will not effect this. The shape of the resulting graph when V0 is plotted against (S) is called hyperbola curve (Fig 10.2).


Fig. 10.2 Effect of substrate concentration on enzyme-catalyzed reaction

10.5 Effect of pH

Enzymes have an optimum pH (or pH range) at which their activity is maximal. At higher or lower pH, activity decreases. Amino acid side chains in the active site may act as weak acids and bases with critical functions that depend on their maintaining a certain state of ionization, and elsewhere in the protein ionized side chains may play an essential role in the interactions that maintain protein structure. Large deviations in pH (Fig 10.3) lead to denaturation of enzyme protein itself , due to interference with many weak noncovalent bonds maintaining the three dimensional structure . A graph of V0 plotted against pH will usually give a bell shaped curve. Many enzyme have pH optimum of around 6.8 but there is a great diversity in pH optima of enzyme due to different environments in which they are adapted to work.


Fig. 10.3 Effect of pH on enzyme-catalyzed reaction

10.6 Enzyme Concentration

In situation where the substrate concentration is saturating (i.e all the enzyme molecules are bound to substrate) , doubling the enzyme concentration will lead to doubling of V0. This gives a straight line graph when V0 is plotted against enzyme concentration

10.7 Effect of Temperature

Raising the temperature increases the rate of both uncatalyzed and enzyme-catalyzed reactions by increasing the kinetic energy and the collision frequency of the reacting molecules. However, heat energy can also increase the kinetic energy of the enzyme to a point that exceeds the energy barrier for disrupting the noncovalent interactions that maintain the enzyme’s three-dimensional structure. The polypeptide chain then begins to unfold, or denature, with an accompanying rapid loss of catalytic activity. The temperature range over which an enzyme maintains a stable, catalytically competent conformation depends upon—and typically moderately exceeds—the normal temperature of the cells in which it resides. Enzymes from humans generally exhibit stability at temperatures up to 45–55 °C. By contrast, enzymes from the thermophilic microorganisms that reside in volcanic hot springs or undersea hydrothermal vents may be stable up to or above 100 °C.


Fig. 10.4 Effect of temperature on enzyme-catalyzed reaction

10.8 Isoenzymes

Isoenzymes are different forms of an enzyme which catalyze the same reaction, but exhibit different physical or kinetic properties, such as isoelectric point, pH optimum, substrate affinity or effect of inhibitors. Different isoenzymes forms of a given enzyme are usually derived from different genes and often occur in different tissues of body. Functional lactate dehydrogenase are homo or hetero tetramers composed of M and H protein subunits encoded by the LDHA and LDHB genes respectively:

• LDH-1 (4H) - in the heart and RBCs
• LDH-2 (3H1M) - in the reticuloendothelial system
• LDH-3 (2H2M) - in the lungs
• LDH-4 (1H3M) - in the kidneys, placenta and pancreas
• LDH-5 (4M) - in the liver and striated muscle

The five isoenzymes that are usually described in the literature each contain four subunits. The major isoenzymes of skeletal muscle and liver, M4, has four muscle (M) subunits; while H4 is the main isoenzymes for heart muscle in most species, containing four heart (H) subunits. The other variants contain both types of subunits.
Last modified: Thursday, 25 October 2012, 5:46 AM