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Introduction to Enzymes
The following has been excerpted from a very popular Worthington publication which was
originally published in 1972 as the Manual of Clinical Enzyme Measurements. While some of the
presentation may seem somewhat dated, the basic concepts are still helpful for researchers
who must use enzymes but who have little background in enzymology.
Enzyme Concentration
Inordertostudytheeffectofincreasingtheenzymeconcentrationuponthereactionrate,the
substratemustbepresentinanexcessamounti.e.,thereactionmustbeindependentofthe
substrateconcentration.Anychangeintheamountofproductformedoveraspecifiedperiodof
timewillbedependentuponthelevelofenzymepresent.Graphicallythiscanberepresentedas:

Table of Contents
I. Introduction
II. Enzymes and Life Processes
Early Enzyme Discoveries
Chemical Nature of Enzymes
Specificity of Enzymes
Naming and Classification
III. Enzyme Kinetics
Basic Enzyme Reactions
Energy Levels
The Enzyme Substrate Complex
Chemical Equilibrium
Factors Affecting Enzyme Activity

Enzyme Concentration
Substrate Concentration
Inhibitors
Temperature
pH
IV. References
Thesereactionsaresaidtobe"zeroorder"becausetheratesareindependentofsubstrate
concentration,andareequaltosomeconstantk.Theformationofproductproceedsatarate
whichislinearwithtime.Theadditionofmoresubstratedoesnotservetoincreasetherate.In
zeroorderkinetics,allowingtheassaytorunfordoubletimeresultsindoubletheamountof
product.
TableI:ReactionOrderswithRespecttoSubstrateConcentration

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TableI:ReactionOrderswithRespecttoSubstrateConcentration

Order

RateEquation

Comments

zero

rate=k

rateisindependentofsubstrateconcentration

first

rate=k[S]

rateisproportionaltothefirstpowerofsubstrateconcentration

second rate=k[S][S]=k[S]2 rateisproportionaltothesquareofthesubstrateconcentration

second rate=k[S1][S2]

rateisproportionaltothefirstpowerofeachoftworeactants

Theamountofenzymepresentinareactionismeasuredbytheactivityitcatalyzes.The
relationshipbetweenactivityandconcentrationisaffectedbymanyfactorssuchastemperature,
pH,etc.Anenzymeassaymustbedesignedsothattheobservedactivityisproportionaltothe
amountofenzymepresentinorderthattheenzymeconcentrationistheonlylimitingfactor.Itis
satisfiedonlywhenthereactioniszeroorder.
InFigure5,activityisdirectlyproportionaltoconcentrationintheareaAB,butnotinBC.
Enzymeactivityisgenerallygreatestwhensubstrateconcentrationisunlimiting.

Whentheconcentrationoftheproductofanenzymaticreactionisplottedagainsttime,asimilar
curveresults,Figure6.
BetweenAandB,thecurverepresentsazeroorderreactionthatis,oneinwhichtherateis
constantwithtime.Assubstrateisusedup,theenzyme'sactivesitesarenolongersaturated,
substrateconcentrationbecomesratelimiting,andthereactionbecomesfirstorderbetweenB
andC.
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Tomeasureenzymeactivityideally,themeasurementsmustbemadeinthatportionofthe
curvewherethereactioniszeroorder.Areactionismostlikelytobezeroorderinitiallysince
substrateconcentrationisthenhighest.Tobecertainthatareactioniszeroorder,multiple
measurementsofproduct(orsubstrate)concentrationmustbemade.
Figure7illustratesthreetypesofreactionswhichmightbeencounteredinenzymeassaysand
showstheproblemswhichmightbeenounteredifonlysinglemeasurementsaremade.

Bisastraightlinerepresentingazeroorderreactionwhichpermitsaccuratedeterminationof
enzymeactivityforpartorallofthereactiontime.Arepresentsthetypeofreactionthatwas
showninFigure6.Thisreactioniszeroorderinitiallyandthenslows,presumablydueto
substrateexhaustionorproductinhibition.Thistypeofreactionissometimesreferredtoasa
"leading"reaction.True"potential"activityisrepresentedbythedottedline.CurveCrepresents
areactionwithaninitial"lag"phase.Againthedottedlinerepresentsthepotentiallymeasurable
activity.Multipledeterminationsofproductconcentrationenableeachcurvetobeplottedand
trueactivitydetermined.AsingleendpointdeterminationatEwouldleadtothefalseconclusion
thatallthreesampleshadidenticalenzymeconcentration.
Next: Effects of Substrate Concentration

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PDF version of Introduction to Enzymes

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