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ENZYMES• They control metabolism by regulating metabolic reaction rates:
molecules that accelerate or catalyze chemical reactions in cells by breaking old covalent bonds & forming new covalent bonds
• Except for Ribozymes, all enzymes are proteins
• a biological catalyst…• have complex structure (sequence of aa’s) • act only upon a specific substrate (or substrate group)• do not change the energetics of the reaction
ENZYME ACTİON E + S <---> [ES] <---> E + P
enzymes catalyze reactions by lowering the energy of activation (Ea)
WHAT DOES AN ES COMPLEX DO?- holds substrate out of aqueous solution- holds substrate in specific orientation, close to Transition
State to allow reaction to occur- reduces ability of free rotation & molecular collisions with
non-reactive atoms- allows an altered local environment: changes ionic strength,
pH, adds or removes H-bonds to substrate
TERMİNOLOGYMany enzymes require a non-protein component for activity:
cofactor: small inorganic ions... mostly metal ions: Cu (cytochrome oxidase), Mg (kinases), Fe (catalase, peroxidase)
coenzymes: small non-protein but organic compounds Coenzyme A: acyl transfer
Flavins: redox reactionNAD+ (NADP+): redox reactions Vitamins: derivatives of B vitamins (B1, B2, B6, B12), niacin, folic acid, riboflavin
prosthetic group: tightly bound large complex organic molecules, (heme)
Holoenzyme vs apoenzyme (apoprotein)
• active site: portion of enzyme which folds to precisely fit the contours of a substrate via weak electrostatic interactions & facilitates bond reactivity
• allosteric site: a site other than the active site
Isoenzymes
Classification is based on reaction catalyzed so enzymes isolated from different organisms but catalysing same rxn have same number but different amino acid sequence
Even within a single species, there may exist different forms of enzyme catalysing the same reaction. Differences may be: A.acid sequence Some covalent modification 3-D structure
Isoenzyme (isozyme): different variants of the same enzyme having identical functions
PROPERTİES OF ENZYMES AS CATALYSTS-1Catalytic power They may increase reaction rate by as much as 1015-fold
2H2O2 2H2O + O2 Rate (L/mol/s)No catalyst 1 x 10-7
Fe2+ catalyst 56Catalase 4 x 107
Specificity Most enzymes are highly specific to their substrate and
reaction catalysed Bond specificity: e.g peptidase, phosphatase Group specificity: e.g hexokinase Absolute or near-absolute specificity
Stereospecificity: Dehydrogenases catalyst the transfer of hydrogen from the
substrate to a particular side of nicotinamide ring in NAD+ or NADP+
Phenylalanine hydroxylase uses L-Phe not D-Phe Importance of specificity in DNA replication and protein
synthesis proofreading
PROPERTİES OF ENZYMES AS CATALYSTS-2Regulation Allosteric regulation (+/- effectors)
e.g. feedback inhibition Covalent regulation (phosphorylation by ATP-dependent
protein kinases) e.g. Glycogen phosphorylase
Activation of zymogens, which are inactive proenzymese.g. trypsinogen
Amount of enzyme: gene expression enzyme degradation
HOW TO DEFİNE ENZYME ACTİVİTY?
Physical properties of an enzyme most often is measured by relative rate that substrate ---> product
1 unit ACTIVITY= International unit (IU)amount enzyme which converts 1 μmole substrate per
min at 25oC e.g. IU= 10 μmole/min
1 unit SPECIFIC ACTIVITY# IU of enzymatic activity per mg of total protein present e.g. 10 μmole/min/mg protein or 10 IU/mg protein
CLASSİFİCATİON OF ENZYMES
Enzyme Commission (EC, 1955) - IUBMB International Union of Biochemistry & Molecular Biology
4 digit Numbering System [1.2.3.4]
1st one of the 6 major classes of enzyme activity2nd the subclass (type of substrate or bond cleaved)3rd the sub-subclass (group acted upon, cofactor required,
etc...)4th a serial number… (order in which enzyme was added to
list)
MAJOR CLASSES OF ENZYMES-11. Oxidoreductases [dehydrogenases, oxidases, peroxidases]
oxidation-reduction reactions, often using coenzyme as NAD+/FAD
Alcohol dehydrogenase [EC 1.1.1.1] CH3CH2OH + NAD+ ---> CH3CHO + NADH + H+
2. Transferases [kinase, phosphorylase, transaminases] group transfer reactions (AX + B BX + A)
Hexokinase [EC 2.7.1.2] D-glu + ATP ---> D-glu-6-P + ADP
3. Hydrolases [digestive enzymes; amylases, lactase, sucrase] hydrolytic reactions: (AX + H2O XOH +
HA) Alkaline phosphatase [EC 3.1.3.1] R-PO4 + H2O ---> R-OH + H-PO4
MAJOR CLASSES OF ENZYMES-24. Lyases [decarboxylases] elimination rxns in which a
double bond is formed Pyruvate decarboxylase [EC 4.1.1.1] pyruvate ---> acetaldehyde + CO2
5. Isomerases [mutases, cis-trans isomerases, racemases] isomerization rxns
Alanine racemase [EC 5.1.1.1] L-alanine ---> D-alanine
6. Ligases [a.acid RNA ligase] condensation of 2 substrates at the expense of energy (ATP)(X + Y + ATP XY + ADP + Pi)
Isoleucine-tRNA ligase [EC 6.1.1.5] L-isoleucine + tRNAIle + ATP ---> L-isoleucyl- tRNAIle + ADP + PPi
MULTİENZYME SYSTEMS Proteins that exhibit more than one catalytic activity EC recommendation more than one catalytic activity
systeme.g. fatty acid synthase system
Multifunctional enzymes will have more than one EC number...
Multifunctional enzyme can made up of: Several polypeptide chains with different catalytic
activities may be associated with each other A single polypeptide chain with multiple catalytic site or even both
Enzymes are used in industrial processes and as analytical reagents in medicine
Immobilisation of enzymes is an important technique used in industry as it enables economical operation of a process and protection of
enzymes during their use
Because of their sensitivity and specificity, enzymes are used as analytical reagents in systems such as the detection of glucose in
human blood and urine
Thermostability and an ability to withstand extremes of pH are
essential properties for enzymes usedin many industrial processes
Enzymes in Biotechnology
Enzyme technology is concerned with the application of enzymesas tools of industry, agriculture and medicine
Enzymes are biological catalysts that fulfil their roleby binding specific substrates at their active sites
This specificity is one property of enzymes thatmakes them useful for industrial applications
The value of using enzymes over inorganic catalysts in the technological field is their efficiency, selectivity and specificity
Enzymes are able to operate at room temperature, atmospheric pressure and within normal pH ranges (around 7)– all of which create energy savings for industry
Enzymes possess specifically shaped active sites for reacting with one specific substrate thereby generating pure products
free from unwanted by-products
Enzymes are biodegradable and, unlike many inorganiccatalysts, cause less damage to the environment
Enzyme Technology
The micro-organisms(such as yeast) are really
used as a source of enzymes during the manufacture of
these products of biotechnology
Many industrial processes now make use of pure sources of enzymes, i.e. the enzymes have been ISOLATED from the micro-organisms before use
Micro-organisms have beenused for thousands of yearsfor making products such as
wine, beer, vinegar, soy sauce,bread and cheese
Products of Enzyme Technology
The large scale production of enzymes involves culturing micro-organismsin chambers called FERMENTERS or BIOREACTORS
Micro-organisms are suitable for use in the large scale production of enzymes in fermenters because:
• They have rapid growth rates and are able to produce larger numbers of enzyme molecules per body mass than many other organisms
• Micro-organisms can be genetically engineered to improve the strain and enhance yields
• Micro-organisms are found in a wide variety of different habitats such that their enzymes are able to function across a range of temperatures and pH
• Micro-organisms have simple growth requirements and these can be precisely controlled within the fermenter
• Micro-organisms can utilise waste products such as agricultural waste as substrates
Large Scale Production of Enzymes
The costs associated with the use of enzymes for industrial purposes can also be reduced by immobilising the enzymes
Enzymes for industrial processes are more valuable when they are able to act in an insolubilised state rather than in solution
Enzymes are immobilised by binding them to, or trapping them in a solid support
Various methods for immobilising enzymes are available
Immobilised Enzymes
Enzymes are held on to a solidsupport (matrix) by weak forcessuch as hydrogen bonding
Enzymes are trapped withinthe structure of a solid polymer(usually in the form of beads)– the enzyme is trapped ratherthan bound
Methods for Immobilising Enzymes
Enzymes are covalently bondedto a matrix such as celluloseor collagen
Another more expensive method involvesenzymes which are both covalently bondedto, and cross-linked within, a matrix
Cross-linking and covalent bonding maycause some enzymes to lose their catalyticactivity especially if the active site is involvedin forming the linkages
Compared with free enzymes in solution, immobilised enzymeshave a number of advantages for use in industrial processes
The stability of many enzymes is increased when they are in an immobilised state; they are less susceptible to changes in
environmental conditions such as temperature and pH fluctuations
Immobilised enzymes can be recovered and re-used,reducing overall costs
The products of the reaction are not contaminated with enzyme eliminating the need to undertake costly separation of
the enzyme from the product
Immobilising enzymes allows for continuous production of a substance with greater automation
Advantages of Immobilising Enzymes
Enzyme Immobilisation and Thermostable Enzymes inThe Production of High Fructose Syrup
This industrial process involves the conversion of cheap corn starch into a high fructose syrup for use as a sweetener in confectionary and drinks
Starch Paste Starch paste is incubated with thethermostable enzyme alpha amylase
at 90oC for a couple of hours
Dextrins(short chains
of glucosemolecules)
Alpha amylase catalyses the hydrolysis of the starchinto short glucose chains called dextrins
The temperature is raised to 140oC to denature theamylase and then lowered to around 55oC before
adding the fungal enzyme amyloglucosidase
Glucose
Amyloglucosidase catalyses the hydrolysis ofdextrins into glucose molecules
Fructose syrup emergesfrom the end of the column
free from contaminationwith enzyme
The final stage involvesthe conversion of glucose
syrup into the much sweeterfructose syrup using the
enzyme glucose isomerase
Glucose isomerase is immobilisedin rigid granules and packed into
a column
Glucose syrup is poured intothe top of the column and ishydrolysed as it contacts the
immobilised enzyme
The sensitivity and specificity of enzymes makes them usefultools in medicine for the detection and measurement of chemicals
in fluids such as blood and urine
Because of their specificity, enzymes will bind to only one substrate – they can therefore be used for the identification
of a specific substance in a biological sample
Because of their sensitivity, enzymes are able to detect thepresence of specific molecules even when they are
present at very low concentrations
The enzyme glucose oxidase is used in an immobilised formfor the detection of glucose in biological fluids
Enzymes as Analytical Agents
The colour of the pad on the clinistix is compared witha colour chart to determine the amount of glucose
present in the sample
Increasing amounts of glucoseNoglucose
Glucose Measurement using 'Clinistix'
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