LAB Topic 3: ENZYMES

Principles of Biology Lab I

Enzymes
Most chemical reactions in cells are too slow to sustain the cellular metabolism Enzymes are biological catalysts that speed up chemical reactions in the cells without being consumed Most enzymes are proteins although some called ribozymes are made of RNA

Enzymes speed the reactions by lowering the activation energy EA

Enzymes cannot decrease or increase the free energy change G of the reactions. We can also say that the enzymes do not alter the equilibrium or the ratio between the initial reagents and final products of the reactions they catalyze Enzymes only speed the reactions toward their equilibrium

Fig. 8-15

Course of reaction without enzyme

EA without enzyme

EA with enzyme is lower

Reactants Course of reaction with enzyme G is unaffected by enzyme

Products Progress of the reaction

Enzymes are very specific

What accounts for this specificity? The specificity of the enzymes results from their unique three dimensional macromolecular structure

Substrate Specificity of Enzymes

The reactant that an enzyme acts on is called the enzymes substrate The enzyme binds to its substrate, forming an enzyme-substrate complex The active site is the region on the enzyme where the substrate binds Induced fit of a substrate brings chemical groups of the active site into positions that enhance their ability to catalyze the reaction
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Catalysis in the Enzymes Active site

In an enzymatic reaction, the substrate binds to the active site of the enzyme The active site can lower an EA barrier by
Orienting substrates correctly Straining substrate bonds Providing a favorable microenvironment Covalently bonding to the substrate

Enzyme activity
The activity of an enzyme or how efficiently it can catalyze a reaction is affected by environmental factors such as Temperature pH Inorganic or organic molecules - cofactors (such as metal ions) or coenzymes (such as vitamins), which are organic molecules Enzyme inhibitors

Enzyme Inhibitors
Competitive inhibitors bind to the active site of an enzyme, competing with the substrate Noncompetitive inhibitors bind to another part of an enzyme, causing the enzyme to change shape and making the active site less effective Examples of enzyme inhibitors include toxins, poisons, pesticides, and antibiotics
Copyright 2008 Pearson Education, Inc., publishing as Pearson Benjamin Cummings

 There are two types of amylases: salivary amylase hydrolyzes starch and glycogen in the food to oligosaccharides; pancreatic - amylase in small intestines continues the digestion of the oligosaccharides to maltose and oligosaccharides called dextrins. Maltose and dextrins are hydrolyzed to glucose on the surface of the intestinal epithelial cells. Glucose enters the blood stream and is used to feed the cells.

During this lab you will study the activity of the enzyme amylase from your saliva

You will perform enzymatic assays to study amylase activity

In an enzymatic assay a sample containing the studied enzyme (for example urine, blood or environmental probe) is incubated with a known substrate of the enzyme and the transformation of the substrate into product is monitored over the time. This is done either by measuring the amount of the accumulated product or by the substrate disappearance during the course of the reaction. The later will be used in this lab

What are you measuring?

You will investigate the -amylase activity by measuring the amount of starch, which is degraded during incubation with mouth rinse sample containing amylase, using Lugol reagent Such measurements are also called fixed time point assays because the disappearance of the substrate is not measured continuously but after a fixed incubation time 10 minutes in your experiment. They are an important tool used in research, clinical and industrial laboratories