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 aas)
act only upon a specific substrate (or substrate group)
do not change the energetics of the reaction

ENZYMEACTON
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 nonreactive atoms
allows an altered local environment: changes ionic strength, pH,
adds or removes H-bonds to substrate

TERMNOLOGY

Many 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 reaction
NAD+ (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

PROPERTES OF ENZYMES AS
CATALYSTS-1

Catalytic power
They may increase reaction rate by as much as 10 15-fold
2H2O2 2H2O + O2 Rate (L/mol/s)
No catalyst 1 x 10-7
Fe2+ catalyst 56
Catalase
4 x 107

Specificity
Most enzymes are highly specific to their substrate and reaction
catalysed

Stereospecificity:

Bond specificity: e.g peptidase, phosphatase

Group specificity: e.g hexokinase
Absolute or near-absolute specificity

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

PROPERTES OF ENZYMES AS
CATALYSTS-2
Regulation
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 proenzymes
e.g. trypsinogen
Amount of enzyme:

gene expression
enzyme degradation

HOW TO DEFNE ENZYME ACTVTY?

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 mole substrate per min at
25oC
e.g. IU= 10 mole/min

1 unit SPECIFIC ACTIVITY

# IU of enzymatic activity per mg of total protein present
e.g. 10 mole/min/mg protein or 10 IU/mg protein

CLASSFCATON 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 activity

2nd 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-1

1.

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-2

4.

Lyases [decarboxylases]
bond is formed

elimination rxns in which a double

Pyruvate decarboxylase [EC 4.1.1.1]

---> acetaldehyde + CO2

5.

Isomerases [mutases, cis-trans isomerases, racemases]

isomerization rxns
Alanine racemase [EC 5.1.1.1]
alanine

6.

pyruvate

Ligases [a.acid RNA ligase]

the expense of energy (ATP)
(X + Y+ ATP

L-alanine ---> D-

condensation of 2 substrates at

XY + ADP + P i)

Isoleucine-tRNA ligase [EC 6.1.1.5]

L-isoleucine +
tRNAIle + ATP ---> L-isoleucyl- tRNAIle + ADP + PPi

MULTENZYME SYSTEMS

Proteins that exhibit more than one catalytic activity

EC recommendation more than one catalytic activity
system
e.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

EnzymesinBiotechnology
Enzymesareusedinindustrialprocessesandasanalytical
reagentsinmedicine

Thermostabilityandanabilitytowithstand
extremesofpHare
essentialpropertiesforenzymesused
inmanyindustrialprocesses

Immobilisationofenzymesisanimportanttechniqueusedinindustry
asitenableseconomicaloperationofaprocessandprotectionof
enzymesduringtheiruse

Becauseoftheirsensitivityandspecificity,enzymesareusedas
analyticalreagentsinsystemssuchasthedetectionofglucosein
humanbloodandurine

EnzymeTechnology
Enzymetechnologyisconcernedwiththeapplicationofenzymes
astoolsofindustry,agricultureandmedicine
Enzymesarebiologicalcatalyststhatfulfiltheirrole
bybindingspecificsubstratesattheiractivesites
Thisspecificityisonepropertyofenzymesthat
makesthemusefulforindustrialapplications
Thevalueofusingenzymesoverinorganiccatalystsinthe
technologicalfieldistheirefficiency,selectivityandspecificity
Enzymesareabletooperateatroomtemperature,atmospheric
pressureandwithinnormalpHranges(around7)
allofwhichcreateenergysavingsforindustry
Enzymespossessspecificallyshapedactivesitesforreactingwith
onespecificsubstratetherebygeneratingpureproducts
freefromunwantedby-products
Enzymesarebiodegradableand,unlikemanyinorganic
catalysts,causelessdamagetotheenvironment

ProductsofEnzymeTechnology
Micro-organismshavebeen
usedforthousandsofyears
formakingproductssuchas
wine,beer,vinegar,soysauce,
breadandcheese

Themicro-organisms
(suchasyeast)arereally
usedasasourceofenzymes
duringthemanufactureof
theseproductsof
biotechnology

Manyindustrialprocessesnowmakeuseofpuresourcesofenzymes,i.e.
theenzymeshavebeenISOLATEDfromthemicro-organismsbeforeuse

LargeScaleProductionofEnzymes
Thelargescaleproductionofenzymesinvolvesculturingmicro-organisms
inchamberscalledFERMENTERSorBIOREACTORS
Micro-organismsaresuitableforuseinthelargescaleproductionof
enzymesinfermentersbecause:
Theyhaverapidgrowthratesandareabletoproducelargernumbersof
enzymemoleculesperbodymassthanmanyotherorganisms
Micro-organismscanbegeneticallyengineeredtoimprovethestrainand
enhanceyields
Micro-organismsarefoundinawidevarietyofdifferenthabitatssuchthat
theirenzymesareabletofunctionacrossarangeoftemperaturesandpH
Micro-organismshavesimplegrowthrequirementsandthesecanbe
preciselycontrolledwithinthefermenter
Micro-organismscanutilisewasteproductssuchasagriculturalwaste
assubstrates

ImmobilisedEnzymes
Thecostsassociatedwiththeuseofenzymesforindustrial
purposescanalsobereducedbyimmobilisingtheenzymes

Enzymesforindustrialprocessesaremorevaluablewhen
theyareabletoactinaninsolubilisedstateratherthaninsolution

Enzymesareimmobilisedbybindingthemto,ortrapping
theminasolidsupport

Variousmethodsforimmobilisingenzymesareavailable

MethodsforImmobilisingEnzymes

Enzymesareheldontoasolid
support(matrix)byweakforces
suchashydrogenbonding

Enzymesaretrappedwithin
thestructureofasolidpolymer
(usuallyintheformofbeads)
theenzymeistrappedrather
thanbound

Enzymesarecovalentlybonded
toamatrixsuchascellulose
orcollagen

Anothermoreexpensivemethodinvolves
enzymeswhicharebothcovalentlybonded
to,andcross-linkedwithin,amatrix
Cross-linkingandcovalentbondingmay
causesomeenzymestolosetheircatalytic
activityespeciallyiftheactivesiteisinvolved
informingthelinkages

AdvantagesofImmobilisingEnzymes
Comparedwithfreeenzymesinsolution,immobilisedenzymes
haveanumberofadvantagesforuseinindustrialprocesses
Thestabilityofmanyenzymesisincreasedwhentheyareinan
immobilisedstate;theyarelesssusceptibletochangesin
environmentalconditionssuchastemperatureandpHfluctuations
Immobilisedenzymescanberecoveredandre-used,
reducingoverallcosts
Theproductsofthereactionarenotcontaminatedwithenzyme
eliminatingtheneedtoundertakecostlyseparationof
theenzymefromtheproduct
Immobilisingenzymesallowsforcontinuousproduction
ofasubstancewithgreaterautomation

EnzymeImmobilisationandThermostableEnzymesin
TheProductionofHighFructoseSyrup

Thisindustrialprocessinvolvestheconversionofcheapcornstarchintoa
highfructosesyrupforuseasasweetenerinconfectionaryanddrinks

StarchPaste

Starchpasteisincubatedwiththe
thermostableenzymealphaamylase
at90oCforacoupleofhours

Alphaamylasecatalysesthehydrolysisofthestarc
intoshortglucosechainscalleddextrins

Dextrins Thetemperatureisraisedto140 Ctodenaturethe

(shortchains amylaseandthenloweredtoaround55 Cbefore
ofglucose addingthefungalenzymeamyloglucosidase
molecules)
o

Amyloglucosidasecatalysesthehydrolysisof
dextrinsintoglucosemolecules

Glucose

Thefinalstageinvolves
theconversionofglucose
syrupintothemuchsweeter
fructosesyrupusingthe
enzymeglucoseisomerase

Glucoseisomeraseisimmobilised
nrigidgranulesandpackedinto
acolumn

Glucosesyrupispouredinto
thetopofthecolumnandis
hydrolysedasitcontactsthe
immobilisedenzyme

Fructosesyrupemerges
fromtheendofthecolumn
freefromcontamination
withenzyme

EnzymesasAnalyticalAgents
Thesensitivityandspecificityofenzymesmakesthemuseful
toolsinmedicineforthedetectionandmeasurementofchemicals
influidssuchasbloodandurine

Becauseoftheirspecificity,enzymeswillbindtoonlyonesubstratetheycan
thereforebeusedfortheidentification
ofaspecificsubstanceinabiologicalsample

Becauseoftheirsensitivity,enzymesareabletodetectthe
presenceofspecificmoleculesevenwhentheyare
presentatverylowconcentrations

Theenzymeglucoseoxidaseisusedinanimmobilisedform
forthedetectionofglucoseinbiologicalfluids

GlucoseMeasurementusing'Clinistix'
Thecolourofthepadontheclinistixiscomparedwith
acolourcharttodeterminetheamountofglucose
presentinthesample

No
glucose

Increasingamountsofglucose