Not all enzymes break down substrates, as in digestion. Some build up polymers, or carry out different sorts of molecular reconstruction.
[See categories below]
An enzyme can be described as a biological catalyst
in that it speeds up (or actually initiates) a biochemical reaction, and remains unchanged at the end of the reaction
The term substrate
is used for reactants in enzyme-controlled reactions.
Enzymes effectively control biochemical reactions.
So as to emphasise this, an enzyme-controlled biochemical reaction is usually written with the enzyme name over the arrow:
substrate(s) → product(s)
An enzyme catalyses the reaction by physically binding with and briefly chemically combining with the the substrate, forming an enzyme substrate complex, which then is changed into products and quickly released.
The enzyme-substrate complex is an efficient way of achieving the chemical change resulting in the product of the reaction and it effectively makes the transformation happen by lowering the activation energy, compared with the uncatalysed reaction.
E+S → ES complex → EP complex → E+P
Or, if you want an animated version:
Complementary means fitting together in a harmonious way;
Complimentary means praising or approving, or free of charge
An enzyme is usually a globular protein with a pocket-like section - the active site - on its surface. This has a particular 3-dimensional shape which is broadly complementary to the substrate so that the substrate molecule (or part of it) fits inside the active site. This is a consequence of the tertiary level of protein structure.
The active site provides an environment where the substrate can become attached, perhaps holding it by hydrogen bonds or other mechanisms, and as a result the enzyme changes shape slightly to become moulded round the substrate. As a result of this closer contact, the enzyme brings to bear reactive groups (amino acid sidechains or co-enzymes) which cause changes in the substrate, effectively weakening bonds and bringing in other groups to make the product.
Sometimes there is mention of binding sites as distinct from the active site where the chemical change is finalised.
As described above, enzymes only work if the substrate fits into the active site. Another substance, with a different molecular shape, will not fit into the active site so it will not be changed by the enzyme.
Factors affecting the rate of enzyme controlled reactions
It is normal to consider the interaction of enzyme and substrate in the context of these molecules being in motion and occasionally forming enzyme-substrate complexes as a result of molecular collisions, but it must be borne in mind that part of the process involves the substrate becoming aligned to bind with the enzyme and then to fit into and react with the active site.
Measuring the rate of enzyme-controlled reactions
In a simple investigation, it is convenient to use an indicator with a well-defined end point.
Timing the change of this effectively gives the rate of disappearance of substrate or the rate of appearance of product.
phenolphthalein in the action of lipase on fats - timing the fall in pH as fatty acids are released
iodine solution - timing the disappearance (conversion) of starch under the action of amylase.
In these cases, the rate of reaction is inversely proportional to the time taken to reach a conversion - 1/T - but the units are rather arbitrary as it is just a qualitative change.
To obtain a more satisfactory and quantitative value for the 'rate of reaction' as shown on these graphs, a series of reaction vessels must be set up, varying one of the following factors and keeping the others constant:
- enzyme concentration
- substrate concentration
- temperature (using a waterbath)
- pH (using a buffer solution)
- (inhibitor concentration)
At appropriate time intervals, samples are taken from the reaction vessel and the (decreasing) concentration of substrate or the (increasing) concentration of product is immediately measured.
A procedure using a spectophotometer is frequently used for this. Often the reagent used to give colour 'quenches' or stops the reaction so that each sample represents a 'snapshot' of the reaction.
The rate of reaction is highest at the beginning of the reaction, and using a graph this initial rate of reaction (also sometimes called the initial velocity, Vi) can be found by taking the slope of the tangent to the change in concentration graph at time t=0.
Depending on the relative concentrations of substrate and enzyme, there will be a varying proportion of enzyme available for reaction, compared with the concentration of enzyme with active site currently in use, but during the course of the reaction the total amount of these forms remains the same. The reaction will proceed at a maximum rate when all the enzyme active sites are in use - 'saturated'.
, there will be a decreasing amount of substrate available for reaction and an increasing amount of product. In other words, the concentration of substrate falls and the rate of reaction falls as the substrate becomes more dilute.
Varying enzyme concentration
When there is an excess of substrate, increasing the concentration of enzyme will result in a faster rate of reaction, so rate of reaction is proportional to enzyme concentration. However the rate of reaction graph will reach a plateau when there is an excess of enzyme.
Varying substrate concentration
Obviously a greater concentration of substrate results in a higher rate of reaction, up to the point when enzyme active sites are saturated.
This graph shows the effect of varying substrate concentration - at two different enzyme concentrations
Some of the following factors are directly related to the protein nature of enzymes.
Concentration of competitive and non-competitive inhibitors
have a molecular structure which is similar to the
substrate, or at least that part which interacts with the enzyme's active site.
The inhibitor occupies the active site instead of the real substrate, thereby preventing normal conversion of substrate into product.
Presumably it does not become chemically converted and eventually leaves the active site.
Click to see the following:
normal enzyme action
reset to start
However by increasing the concentration of substrate it is possible to replace the inhibitor with substrate, effectively out-competing the inhibitor.
This graph shows the different effects of competitive and non competitive inhibitors interacting with varying substrate concentrations
have a different molecular structure to the substrate, so they do not combine with the active site. Instead they combine - usually by covalent bonding - with a different area of the enzyme molecule and change the shape of the active site, so that enzyme conversion does not take place.
This is called an allosteric effect and the region on the enzyme where the inhibitor attaches is called an allosteric site.
In this case, adding extra substrate does not result in an increase in rate of reaction.
Small changes in pH can affect charges on acidic and basic sidechains of amino-acids, especially those near to active sites. This can lead to reduced binding of substrate and hence a slower rate of reaction. This is reversible if the pH returns to normal.
Greater changes in pH can be more damaging and irreversible.
Three different digestive enzymes, each with a different optimum pH
Many enzymes have an optimum pH near to 7, especially those working within cells (intracellular enzymes). Others - usually extracellular enzymes - work best above or below this.
Within the digestive system different processes necessitate different conditions. For example it is suggested that in the stomach, digestion of proteins is optimised by the acid-generated unfolding of polypeptide chains, allowing access by proteolytic enzymes. Elsewhere, the digestion of lipids is assisted by bile salts which emulsify fats and oils in alkaline conditions, and the appropriate enzymes have correspondingly different pH optima.
For example, the proteolytic enzyme pepsin has an optimum pH of 2.0 which corresponds with normal stomach conditions, and trypsin (also proteolytic) has an optimum pH of 7.8-8.7 which is normally found in the ileum. Pancreatic lipase, active in the same area, has an optimum pH of 8.0.
When testing for the action of other factors, it is normal to include a pH buffer so as to minimise the effect of pH. Sometimes the chemical components of buffers have a side effect on the reaction, so they must be chosen with caution. However it must be remembered that some change must be expected if the enzyme-controlled reaction actually generates acids or alkalies, and this may exceed the buffering capacity of the actual mixture used. An example is hydrolysis of lipids (producing fatty acids).
It might be sufficient to measure the pH (using a pH meter) at the beginning and end of the reaction, to check there is no significant change.
Most enzymes have a well-defined peak activity at a set temperature - the optimum temperature. This peak is a consequence of two factors; one 'positive', and the other 'negative'.
The two sides of the peak for this graph have different gradients so it is not symmetrical
Below this value, the activity depends on kinetic energy of molecules, which increases by a factor of 2 for each 10 °C rise in temperature. Above it, the enzyme's activity falls markedly as the enzyme itself becomes denatured due to heat weakening hydrogen bonds so that the shape of the active site becomes permanently altered.
This change (denaturation) may take some time to occur in laboratory experiments; enzyme mixed with substrate and then placed in a waterbath at a set (range of) temperature may appear to be much more active than a mixture of substrate with enzyme which has been exposed for some time to the same temperature.
Do not assume that all enzymes have an optimum temperature of 37 ° C; This is not even true for all human enzymes, and lots of enzymes are obtained from micro-organisms which are active at much higher temperatures.
Using a tangent to find the initial rate of an enzyme-controlled reaction
This graph is taken from a specimen paper question.
The blue and red lines have been drawn touch the curves at time t=0 and can be drawn to any length.
In this case the gradient can be easily calculated by dividing the amount of substrate converted (concentration of product in g per dm3
) : y-axis by the time in minutes : x-axis.
At 37 °C the rate of reaction is 10/10 = 1.0 (units g.dm-3
. min -1
At 60 °C the rate of reaction is 10/2.2 = 4.54 (g.dm-3
. min -1
In other words the rate of reaction of this enzyme is 4.54 times greater for a temperature increase of 23 °C
Models of enzyme action
The word enzyme means "in yeast" as some of the early work centered on the fermentation process - responsible for production of wine, beer etc. Louis Pasteur, who showed that living micro-organisms (yeasts) participated in the production of fermented drinks whilst others (Acetobacter
spp) caused spoilage by converting alcohol (ethanol) into acetic acid (ethanoic acid). However he believed that in these processes living cells were necessary, contributing a vital force - a "ferment" - rather than any chemical products of their activity. This was the basis of an ongoing dispute with Justus von Liebig from 1859 to 1869. Eduard Büchner showed by filtration that cells themselves were not necessary and hypothesized that the fermentation was caused by an enzyme which he named zymase in 1897.
When John Northrop showed in 1929 that pepsin could be purified and crystallised he went on to show that it was a protein, thus disbunking the idea that enzymes belonged to a special and unknown class of compounds. He obtained a share in the Nobel Prize for Chemistry in 1946.
Emile Fischer had proposed in 1890 that enzyme and substrate interact like a lock and key
. The substrate being the only substance that will fit into the enzyme to be transformed into product. This approach attempted to explain the specificity of enzymes - each enzyme will only interact with one substrate or type of substrate just as only the correct key will fit into a lock and unlock a door.
The shape of different molecules is in fact central to many concepts in Biological chemistry, most of which involve complementary shapes: antibody-antigen and hormone-receptor reactions as well as DNA and RNA interconversions.
The most important part of an enzyme is seen as the active site - into which the substrate fits, to be converted into the product.
In 1958 Daniel Koshland introduced a refinement to this concept: the enzyme changing shape as it interacts with the substate - the induced fit
model. This takes into account the flexibility of the enzyme's protein structure as well as the interaction with amino acid groups involved in the conversion process.
From the 1960's onwards, the details of the molecular structures of a number of enzymes have been elucidated and their three-dimensional shapes have confirmed the significance of the active site concept. Furthermore, details of protein structure near to this have shown that binding of substrates next to specific amino acid sidechains (and co-enzymes) offers ways of explaining the lower activation energy for the enzyme-controlled reaction compared with the alternative reaction, not involving enzymes.
Enzymes and energy
A chemical reaction involves a change:
reactant(s) → product(s)
In thermodynamic terms, the product(s) have a lower [chemical potential] energy than the reactant(s). In other words the reaction results in the loss of some of this energy (usually as heat) into the environment - an exergonic reaction, sometimes unhelpfully referred to as a spontaneous reaction. However the reaction does not necessarily occur without some form of assistance, which involves the input of extra energy: activation energy
. This effectively acts as an energy barrier to the reaction.
Chemists often get over this by providing heat energy, pressure or an electrical discharge.
However in biological systems the temperature normally remains fairly cool and stable.
An enzyme is said to lower the activation energy for the reaction it controls. It does not alter the initial or final energy levels of the reaction. In essence an enzyme offers a different reaction mechanism/pathway, involving small changes which collectively result in the conversion of reactant into product.
Co-enzymes and co-factors
A coenzyme is a (non-protein) substance with a fairly small molecule that binds with an enzyme - at or near the active site - and assists with the conversion. Typically, it brings in components to be used in the enzymic conversion, such as acting as an electron carrier, or providing (or removing) specific atoms or functional groups that are transfered in the overall reaction, but they are not considered to be substrates as such.
may be considered as a coenzyme.
Many coenzymes are derived from water-soluble (mainly B-group) vitamins.
, Coenzyme A, Coenzyme Q.
A co-factor is usually a metallic ion, which is required for, or increases the rate of, catalysis.
Enzymes and evolution
All enzymes are produced inside the cells of living organisms.
As such, they have been subject to selection pressure in the same way that living organisms face in ordinary life.
The fact that they work so efficiently can be seen as part of the 'survival of the fittest'.
Biochemical mistakes - for good and bad
There are many examples of inherited conditions (in humans and other species) which can be traced back to a modified version of a gene, resulting in the production of a form of an enzyme which does not function as efficiently as normal, or is absent. These 'inborn erros of metabolism' have contributed significantly to our knowledge of genetics.
Nowadays it is possible to make modified ('mutant') forms of proteins in the laboratory by changing bases within the DNA of genes which code for enzymes, so that they contain different amino acids, usually altering only one amino acid at a time. If the resulting modified versions of enzymes are not as active as the original it often shows which parts of the enzyme molecule are functional.
Drugs and poisons
Some (medical) drugs achieve their effects by inhibiting the action of (overactive or unwanted) enzymes in the body.
Aspirin - acetylsalicylic acid - acts as an irreversible inhibitor of the cyclooxygenase (COX-1) enzyme in platelets and thus reduces the likelihood of thrombosis in the blood circulation.
Paracetamol reduces the activity of another cyclooxygenase enzyme (COX-2) which is required for the synthesis of prostaglandins.
NSAIDs (non-steroidal anti-inflammatory drugs) e.g. Ibuprofen similarly reduce inflammation and pain by inhibiting COX-2
The enzyme xanthine oxidase in the liver can convert xanthine, a purine base derived from guanine, into uric acid, which passes into the blood stream and then builds up as painful deposits, typically in toes and fingers, on account of its low solubility. This condition is known as gout.
is a competitive inhibitor of xanthine oxidase and can be used to relieve the symptoms of gout.
Penicillin and other β-lactam antibiotics kill some types of bacteria (Gram-positive bacteria) by interfering with bacterial cell wall synthesis. The four-membered β-lactam 'ring' of these compounds binds to the to the bacterial enzyme DD-transpeptidase, preventing the formation of peptidoglycan cross-links in the bacterial cell wall;
Protease inhibitors are a class of antiviral drugs that are widely used to treat high profile viral diseases HIV/AIDS and hepatitis C. They selectively bind to viral protease enzymes (e.g. HIV-1 protease) which cause cleavage of protein precursors which are necessary for the production of infectious viral particles. Saquinavir was the first of several antiretroviral drugs, all with names ending -avir. This was approved in 1995, and it has resulted in dramatic reduction in deaths from AIDS.
The cyanide ion CN-
is a non-competitive inhibitor of the enzyme Cytochrome C oxidase (also known as aa3) in the electron transport chain (found in the membrane of the mitochondria of eukaryotic cells) and it prevents aerobic respiration. It attaches to the iron within this protein, preventing transport of electrons from Cytochrome C to oxygen. As a result, the cell can no longer aerobically respire to produce ATP for energy-requiring processes.
The heart and central nervous system are particularly affected.
Nerve gases or nerve agents, e.g. sarin, tabun, are a class of phosphorus-containing organic chemicals (organophosphates) - originally developed as insecticides. They work by inhibiting the enzyme acetylcholinesterase, which catalyzes the breakdown of acetylcholine, a neurotransmitter. Normally this is quickly broken down after a nervous impulse passes from one neurone to another, but the effect of breathing in nerve agents is loss of control over respiratory muscles and other bodily functions.
There are six broad categories:
- 1 Oxidoreductases: catalyse oxidation/reduction reactions
- 2 Transferases: transfer a functional group (e.g. a methyl or phosphate group)
- 3 Hydrolases: catalyse the hydrolysis of various bonds
- 4 Lyases: cleave various bonds by means other than hydrolysis and oxidation
- 5 Isomerases: catalyse isomerization changes within a single molecule
- 6 Ligases: join two molecules with covalent bonds
Following on from this, enzymes are categorised by being given an EC (Enzyme Commission) number, with 4 numbered sections, progressively finer degrees of distinction.
EC 3.2 covers enzymes that hydrolyse glycosyl compounds (glycosylases) (!)
EC 3.2.1 includes several enzymes which could be called carbohydrases. Within this:
EC 22.214.171.124 is α-amylase
EC 126.96.36.199 is maltase
EC 188.8.131.52 is invertase (sucrase)
However this is actually based on the chemical reaction catalysed rather than specific biological features such as the 'donor' organism or the part of the body.
Thus salivary amylase shares the same EC number (184.108.40.206) with pancreatic amylase.