San Beda College of Medicine MendiolaBiochemistry

Module 2: EnzymesObjectives

1. To know the distinguishing properties of enzymes.

2. To know what is enzyme kinetics.3. To identify the different types of

enzyme inhibition.4. To know the mechanisms of enzyme

action.5. To know how are enzymes

regulated.6. To know how are enzymes used in

clinical diagnosis.

ENZYMES Biologic polymers that catalyze the chemical

reactions that make life as we know it possible.

Protein catalysts (except for RNA molecules or ribozymes).

Catalytic activity alters speed of chemical reactions

Neither consumed nor permanently altered after during a reaction.

lowers free energy of activation Stabilizes high energy intermediates

ENZYME STRUCTURES Holoenzyme: The complete, active enyzme . The holoenzyme has a protein portion

(APOENZYZME) and a COFACTOR A tightly bound cofactor is called a

PROSTHETIC GROUP

ENZYME NOMENCLATURE The commonly used names for most enzymes

described the type of reaction catalyzed, Followed by the suffix –ase.o eg. dehydrogenase : removes hydrogen

atomo isomerases : catalyze rearrangement in

configuration.Other ways of naming enzymes:

Based on the source of the enzymeo eg. Pancreatic ribonuclease

Based on the mode of regulationo eg. Hormone-sensitive lipase

Based on the distinguishing characteristic of their mechanism

o eg. Serine protease

INTERNATIONAL UNION OF BIOCHEMISTS (IUB System) Unambiguous system of enzyme nomenclature. Each enzyme has a unique name and code

number that identify the type of reaction catalyzed and the substrates involved.

Removes ambiguity and confusiono Eg. Common name : Hexokinase

IUB -ATP:D-hexose-6-phosphotransferase E.C. 2.7.1.1

IUB Enzyme NomenclatureE.C. 2.7.1.1

• First digit – class 2: transferase • Second digit – subclass 7: transfer of

phosphoryl group• Third digit – sub-subclass 1: alcohol is

phosphoryl acceptor• Fourth digit – for specific enzyme

SIX IUB CLASSES OF ENZYMES1. Oxidoreductases

o catalyze oxidation-reduction reactions

2. Transferases o Catalyze transfer of group from donor

compound to acceptor.

3. Hydrolases o Catalyze hydrolysis of bonds.

4. Lyases o Catalyze cleavage of C – C, C – O, C-N

bonds by means other than hydrolysis or oxidation; group elimination to form double bonds.

5. Isomerases o catalyze intramolecular rearrangements.

6. Ligases o catalyze joining of two molecules coupled

with the hydrolysis of a pyrophosphate bond in ATP or similar triphosphate .

ENZYME KINETICS Field of biochemistry concerned with

quantitative measurement of the rates of enzyme-catalyzed reactions and the systematic study of factors that affect these rates.

Important in understanding how stress affects homeostasis.

Enzyme-catalyzed reactions are 103 – 1011 faster than uncatalyzed reactions.

Enzymes do this by decreasing the free energy of activation.

• Activation energy o Amount of energy required to bring the

reactants to the transition state. Gibbs free energy (ΔG)

o Describes both direction in which a chemical reaction will tend to proceed and the concentration of reactants and products that will present equilibrium.

o The ΔG for a chemical reaction equals the sum of free energies of formation of the reaction products ΔGp minus the sum of free energies of formation of substrates ΔGs.

• Standard free energy change (ΔGo) o The difference between the energies of

the reactant (initial state) and the energies of the products (final state).

o If the free energy formation of formation of the products is lower than that of the substrates, the signs of ΔG° will be negative meaning that there’s a favoured reaction in the direction from left to right.

o This reaction is referred to as spontaneous reaction.The formula for ΔG° is:

ΔG°= -RTlnKeq

Where;[R] Gas constant (1.98 cal/mol°k or 8.31 J/mol°K)[T] Absolute temperature in Kelvin[Keq] concentrations of of reactions of products divided by the product of substrates raised to the power of their stoichiometry.

o If ΔG° is negative, the concentration of the products at equilibrium will exceed than that of substrates.

o If ΔG° is positive, the formation of the substrates will be favoured.

MICHAELIS MENTEN EQUATIONo Expresses velocity as a function of

substrate concentration.

Where;[S] Substrate concentration

[Vmax] maximum velocity maximal number of subtrate molecules converted to product per unit time.[Km] Michaelis constant substrate concentration at which Vi is half the maximal velocity (Vmax/2)

o high Km = low substrate affinityo low Km = high substrate affinity

LINEWEAVER-BURK EQUATIONo From Hans Lineweaver and Dean

Burk.o Derived from Michaelis-Menten

equation by simply taking its reciprocal for solving 1/v 0:

v0=Vmax [S]KM+[S ]

1v0

=KM+[S]V max [S ]

1v0

=KMV max

×1

[S ]+ 1V max

or

o Yields a double reciprocal ploto Y intercept is 1/vmax o X intercept is -1/Km

Note:Michaelis Menten Equation yields a hyperbolic curve whereas Lineweaver-Burk will give a straight line.

SUBSTRATE SPECIFICITY• Geometric Complementarity

o Substrate-binding site in enzyme is complementary in shape to the substrate.

• Electronic Complementarity o Amino acid residues that form the

binding site are arranged to interact specifically with the substrate in an attractive manner.

• Stereospecificity o Enzymes are highly specific both in

binding chiral substrates and in catalyzing their reactions

o eg. Enzymes involved in glucose metabolism are specific for D-glucose

“THREE-POINT ATTACHMENT” OF SUBSTRATE TO ENZYME

CATALYTIC TRIAD OF AA:Consists of serine, histidine, and aspartate

1. Histidine bridges serine and aspartate 2. A covalent adduct is formed between the

active site serine and the carbonyl group of a peptide bond, which displaces the amino group from the peptide bond

3. Polypeptide is released form active site ACTIVE SITEo Region of an enzyme concerned with

substrate binding and catalysis.MODELS OF ACTIVE SITE• LOCK AND KEY or RIGID TEMPLATE

MODEL (Fisher)o Catalytic site is presumed pre-shaped to

fit the substrate

• INDUCED FIT MODEL (Koshland)o Binding of the substrate induces a

conformational change in the enzyme that results in a complementary fit once the substrate is bound

COENZYMESo Organic compounds that take part in

enzymatic reactions.o Are chemically changed by the enzymatic

reactions in which they participate; must be returned to their original state in order to complete the catalytic cycle

o Serves as recyclable shuttles or group transfer agents.

o Mostly derived from water-soluble B vitamins.

ISOENZYMES• Physically distinct versions of a given

enzyme, each of which catalyze the same reaction

• Usually are oligomeric enzymes with different protomers in various combinations; one tissue produces one protomer predominantly and another tissue, a different protomer

• Arises from gene duplication.

Clinical applications of enzymes• Measurement of enzymes is used

diagnostically

Some enzymes are used as therapeutic agentso Streptokinase – used to clear clots in the

lower extremities and after a Myocardial Infarction

o Asparaginase – used for the treatment of some types of adult leukemia

Many drugs are enzyme inhibitorso Methotrexate – used in cancer

chemotherapy; inhibits dihydrofolate reductase

o Allopurinol – used in the treatment of gout; inhibits xanthine oxidase

o Statins – used to lower plasma cholesterol; inhibits HMG CoA reductase

o Aspirin – analgesic and anti-inflammatory; inhibits cyclooxygenase, the enzyme for the synthesis of prostaglandins

o Penicillin – antibiotic; inhibits transpeptidase, the enzyme involved in cell wall synthesis.

ENZYME-LINKED IMMUNOASSAYS Used for rapid screening and quantification of

the presence of a protein in a given sample as in enzyme-linked immunosorbent assays (ELISA)

Used to detect proteins that lack catalytic activity.

ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)

Use antibody covalently linked to a “reporter enzyme” such as alkaline phosphatise or horse radish peroxidise.

Use absorbance via light or fluorescence. Used in pregnancy testing.

KINETIC THEORY Also called collision theory.• For two molecules to react, they must:

o Approach within bond-forming distance of one another or “collide”

o Must possess sufficient kinetic energy to overcome the energy barrier for reaching the transition state

• Anything that increases the frequency and the energy of collision between substrates will increase the rate of the reaction

FACTORS THAT AFFECT REACTION RATE1. Temperature

– Increase temperature à increase kinetic energy of molecules à increase motion and frequency with which molecules collide

2. Reactant concentration

– Frequency with which molecules collide is directly proportionate to their concentration

FACTORS THAT AFFECT RATES OF ENZYME-CATALYZED REACTIONS1. TEMPERATURE

– Initially, reaction rate increases as temperature rises (due to increase kinetic energy of reacting molecules)

– Eventually, further increase in temperature breaks hydrogen and hydrophobic bonds that maintains 2o and 3o structure à denaturation à loss of catalytic activity

• Enzymes from humans generally exhibit stability at temperature up to 45o – 55o C

• Q10 or temperature coefficient– Factor by which the rate of a biologic

process increases for a 10oC increase in temperature

• For most biologic processes, Q10 = 2; rate doubles for a 10o C rise

2. pH– Affects enzyme activity by:

• Enzyme denaturation at low or high pH• Alteration in charged state of enzyme

and/or substrate• Change in the charge of group which is

distal to region where substrate bound but which is needed to maintain 3o and 4o structure.

OPTIMUM pH OF SOME ENZYMES

3. SUBSTRATE CONCENTRATION Velocity of reaction increases as concentration of

substrate is increased (first order kinetics), however there will come a point when further increases in substrate will not increase velocity (zero order)

4. ENZYME CONCENTRATION• Initial velocity of reaction is directly

proportional to concentration of enzyme, providing substrate is in excess.

• When the substrate concentration is equal to Km value, the initial velocity (vi) is half- maximal.

Km can be considered an inverse measure of the affinity of the enzyme for the substrate.

The lower the Km, the higher is the affinity.

Question: From the table below, which substrate has a greater affinity for hexokinase?

ENZYME SUBSTRATE Km (mM)

Hexokinase Glucose 0.15

Fructose 1.5 Answer: Glucose because it has a lower Km value than that of Fructose.SIGNIFICANCE OF Km AND Vmax

• Vmax is related to the turnover number of an enzyme, a quantity equal to the catalytic constant kcat .

• Turnover number is the number of moles of substrate that react to form product per mole of enzyme per unit time.

LINEWEAVER – BURK PLOT also know as “DOUBLE RECIPROCAL PLOT” Converts hyperbolic curve to a straight

line

ENZYME INHIBITORS

COMPETITIVE INHIBITORo Resembles the substrate (substrate analogs).o compete for the same binding site on the

enzymeo A mutually exclusive process: when the inhibitor

is bound, substrate is unable to bind and vice versa.

o A competitive inhibitor acts by decreasing the number of free enzyme molecules available to bind substrate.

o Raises the apparent Km (Km’) for the substrate

o At high substrate concentration, Vmax is unchanged.

The diagram above shows the kinetic scheme for competitive inhibition.

Note: A very high concentration of substrate lessens the effect of the competitive inhibitor, unless the inhibitor has a much higher binding activity for enzyme than does the substrate.The inhibitor molecule cannot be converted to a product because it does not have a functional groups normally acted on by the enzyme.

NON-COMPETITIVE INHIBITORo Bears no structural resemblance to substrate.o Binds to a different site on the enzyme.o Both inhibitor and substrate can bind

simultaneously to the enzyme molecule. o Do not affect Km; lowers Vmax.

The diagram above shows the kinetic scheme for noncompetitive inhibition

Note: The presence of the inhibitor does not affect substrate binding but does interfere with the catalytic functioning of the enzyme.The actual mechanism of action for the inhibitor varies with the catalytic functioning of the enzyme.

UNCOMPETITIVE INHIBITOR• Binds only to ES complexes at locations other

than the catalytic site. • Substrate binding modifies enzyme structure,

making inhibitor-binding site available. Inhibition cannot be reversed by substrate.

• Apparent Vmax decreased; Km is decreased.

Note: Uncompetitive is similar to noncompetitive since it allows substrates to bind to the active site however it differs since this type of inhibition binds only to the ES complex.

IRREVERSIBLE ENZYME INHIBITOR• Forms a non-dissociable complex with the

enzymeo Cyanide is a classic example of an

irreversible enzyme inhibitor; inhibits cytochrome oxidase, an enzyme in the respiratory chain

The kinetic effect of irreversible inhibitors is to decrease the concentration of active enzyme, thus decreasing the maximum possible concentration of ES complex.

ENZYME INHIBITORS AS DRUGS• If the drug requirement is to increase the

intracellular concentration of the substrate, either a competitive or non-competitive inhibitor will serve, since both will inhibit the utilization of substrate, so that it accumulates.

• If the drug requirement is to decrease the intracellular concentration of the product, then the inhibitor must be non-competitive.

• As unused substrate accumulates, the increasing substrate concentration overcomes the effect of the competitive inhibitor. Increasing the concentration of substrate does not affect a non-competitive inhibitor.

• For a competitive inhibitor, the lines converge above the x axis, and the value of [I] where they intersect is -Ki

IRREVERSIBLE INHIBITION Forms covalent or a very strong non covalent

bonds with the enzyme.• Forms a very stable complex• Inhibition of cyclooxygenase by aspirin, which

involves acetylation of a serine residue .

DRUGS WHOSE STRUCTURE RESEMBLE SUBSTRATES

• Captopril for treatment of hypertension and congestive heart failure, which inhibit Angiotensin-Converting Enzyme (ACE)

• Simvastatin for hypercholesterolemia, which inhibit HMG CoA Reductase, the rate-limiting enzyme of cholesterol synthesis

BISUBSTRATE REACTIONS• Enzymatic reactions involving two substrates

and yielding two products (“Bi-Bi” reactions)• Both substrates are present at saturating

levels.

• Account for ~ 60% of known biochemical reactions

• Are either: o TRANSFERASE reactions in which

the enzyme catalyzes the transfer of a specific functional group X from one of the substrate to the other

o OXIDATION – REDUCTION reactions in which reducing equivalents are transferred between two substrate

1. SEQUENTIAL REACTIONS

– Reactions in which all substrates must combine with the enzyme before a reaction can occur and products be released

– Also called single displacement reaction because group undergoing transfer is usually passed directly, in a single step, from one substrate to the other

TYPESA. Ordered / Compulsary o compulsory order of substrate addition to the

enzymeo A must first combine with E before B can

combine with EA complex.B. Randomo no preference for order of substrate additiono Either substrate A or substrate B may combine

first with the enzyme to form EA or EB complex.2. PING – PONG REACTIONS One or more products are released before all

substrates have been added. Involve covalent catalysis and a transient,

modified form of enzyme. Enzyme alternates between two forms E and F. Also called double displacement reactions.

CONTROL OF ENZYME AVAILABILITY• Control of enzyme quantity

– Enzyme induction– Enzyme repression– Enzyme turnover

• Enzyme compartmentation • Formation of macromolecular complex• Partial proteolysis• Covalent modification• Regulation by allosteric effectors

ENZYME INDUCTION• Cells can synthesize specific enzymes in

response to the presence of specific inducers• In many instances, the substrate of the

enzyme also serves as inducer

– E.g., lactose inducing the synthesis of b-galactosidase

ENZYME REPRESSION• Curtailment of enzyme synthesis by the

presence in the reaction medium of the metabolite being synthesize (product feedback repression)

o E.g., In S. typhimurium, addition of His represses synthesis of enzymes of histidine synthesis

o Upon removal or exhaustion of the essential intermediate from the medium, enzyme synthesis resumes (derepression)

ENZYME TURNOVER• Combined process of enzyme synthesis and

degradation

• Susceptibility of enzyme to proteolytic degradation:

– Depends upon its conformation which in turn is dependent on the presence or absence of substrate, coenzyme or metal ions

– Also affected by physiologic, hormonal or dietary manipulation

– Examples:o áArginase synthesis – after a protein-rich

dieto â Arginase degradation - during starvationo á Tryptophan oxygenase synthesis –

presence of glucocorticoid o â Tryptophan oxygenase degradation –

presence of tryptophanENZYME COMPARTMENTATION

• Localization of specific metabolic process in the cytosol or in cellular organelles

– E.g., fatty acid oxidation occurs only in the mitocondria; fatty acid synthesis occurs in the cytosol

• Requires “shuttle” mechanisms for translocation of metabolites across compartmental barriers

FORMATION OF MACROMOLECULAR COMPLEX

• Organization of a set of enzyme that catalyze a protracted sequence of metabolic reaction so as to coordinate the enzymes and channel metabolites along metabolic path.

CONTROL OF ENZYME ACTIVITY Partial proteolysis

o Conversion of an inactive proenzyme (zymogen) to a catalytically active form

o Mechanism utilized by proteases and other digestive enzymes and enzymes involved in blood coagulation

o Cleavage at N-terminal between Arg 15 and Ile 16

o Results in the formation of the catalytic site and the substrate-binding pocket.

o Conversion of Chymotrypsinogen to Chymotrypsin

Covalent modification

o Involves phosphorylation of specific serine residues in the enzyme

o Enzymes are phosphorylated or dephosphorylated in response to extracellular signals such as hormones or growth factors

o Enzymes are interconvertible to two activity state – high or low catalytic efficiency

o eg. In muscles,o phosphorylation o activate glycogen o phosphorylase and o inhibits glycogen o synthase o Kinase – catalyze phosphorylation o Phosphatase – catalyze

dephosphorylation

Regulation by allosteric effectorso Involves control of enzyme activity by

binding of a substance (effector) to an allosteric site that is physically distinct from the catalytic site.

o Negative allosteric effector – inhibits the enzyme

o Positive allosteric effector – activates the enzyme

FEEDBACK INHIBITION• Inhibition of activity of an enzyme in a

biosynthetic pathway by an end-product of the pathway

• Form of allosteric inhibition

Multiple Feedback Inhibitions in a Branched Biosynthetic Pathway

1. Cumulative feedback inhibitiono Inhibitory effect of 2 or more end-

products on a single regulatory enzyme is strictly additive

2. Concerted or multivalent feedback inhibitiono Complete inhibition occurs only when

2 or more end-products are present in excess

3. Cooperative feedback inhibitiono A single end-product present in

excess inhibits the regulatory enzyme but inhibition when 2 or more end-products are present far exceeds the additive effects of cumulative feedback inhibition

ALLOSTERIC REGULATION• Allosteric regulation enables a cell to adjust

an enzymatic activity almost instantaneously in response to changes in the concentration of a metabolite since it does not require an intermediate enzyme

• Binding of an effector at an allosteric site usually changes the conformation at the active site

• Enzyme can exist in two conformation:R (relaxed) state or T (tense) state

o Allosteric activator binds preferentially to the R state and stabilizes the conformation that is more effective at binding the substrate

o Allosteric inhibitor acts by stabilizing the T state

Comparison between Covalent Modification and Allosteric Regulation

Similarities:1. Provides for short-term regulation 2. Provides for a reversible means of

regulation 3. Acts without altering gene expression4. Acts on an early enzyme of a

protracted metabolic sequence5. Acts at an allosteric rather than

catalytic site6. Effect is rapid

Differenceso Covalent modification:

Involves several proteins and ATP

Under direct neural and hormonal control

o Allosteric regulation: Involves single protein Lack hormonal and neural

featuresPROXIMITY AND ORIENTATION EFFECT

• The binding of the substrate to the enzyme brings together the substrate molecule and the catalytic group at the active site of the enzyme in perfect arrangement for the given reaction to occur

Immobilization of substrates on an enzyme active site can increase collision probability, juxtaposition of reactive groups and also influence the precise alignment of orbitals needed for the reaction to occur

DESOLVATION EFFECT• In an aqueous medium, the charged groups of

the substrate are neutralized by the dipolar water molecules

• When the substrate becomes embedded in the hydrophobic environment of the active site, the formation of hydrogen bonds and electrostatic bonds with the reacting groups in the enzyme produces polarizing effect on the electrons of the substrate facilitating the reaction

STRAIN EFFECT• A conformational change subsequent to

substrate binding may lead to distortion of some portion of the substrate

Mechanism of action of lysozyme• Lysozyme is an enzyme that ruptures certain

bacterial cells by cleaving the polysaccharide chains that makes up their cell wall.

• Its mechanism of action involves strain-induced destabilization of the sugar when bound to the active site

GENERAL ACID AND GENERAL BASE CATALYSIS• General acid – any substance that is capable

of releasing a proton in an aqueous solution• General base – any substance that is capable

of binding a proton in an aqueous solution• Chemical bonds are formed by electrons

• Formation or breakage of bonds requires the migration of electrons

• Reacting groups either acts as an electron donor or electron acceptor; or as nucleophile or electrophile

• Nucleophile – electron-rich substance that reacts with an electron-deficient substance

• Electrophile – electron-deficient substance that reacts with an electron-rich substance

• The task of a catalyst often is to make a potentially reactive group more reactive by increasing its electrophilic or nucleophilic character

• In many cases, the simplest way to do this is to add or remove a proton

• Proteins contain numerous ionizable groups that can serve as general acid or general base and which can be positioned precisely with respect to the substrate in the active site allowing the proximity effect to come into play

COVALENT CATALYSIS•Involves the formation of an intermediate state

in which the substrate is covalently attached to a nucleophilic group of the enzyme

• - OH group of serine• e-NH2 group of lysine• - SH group of cysteine

MECHANISM OF ACTION OF SERINE PROTEASESSERINE PROTEASES• Includes the digestive enzymes,

chymotrypsin, trypsin and elastase and clotting factor, thrombin

• Uses a highly reactive seryl residue for the formation of a covalent acyl-enzyme intermediate

CHYMOTRYPSIN• Catalyzes hydrolysis of peptide bonds in

which the –COOH group is contributed by an aromatic amino acid (Phe, Tyr, Trp) or by one with a bulky non-polar R group (Met)

• Also catalyzes the hydrolysis of certain esters

• Ser 195 plays an important role in the catalytic mechanism

• Diisopropylfluorophosphate inhibits chymotrypsin by attaching covalently to the serine residue

• Reactivity of Ser 195 is not a property of serine residues in general but has been brought about by its juxtaposition with His 57 and Asp 102

• Forms a “charge-relay network” that functions as a proton shuttle

CHYMOTRYPSIN, TRYPSIN AND ELASTASE• Have similar amino acid sequence, 3-

dimensional structure and catalytic mechanism

• Exhibit very different specificities– Chymotrypsin – acts on peptide

bonds of amino acid with an aromatic ring (Phe, Tyr, Trp)

– Trypsin – acts on peptide bonds of amino acid with positively charged R group (Lys, Arg)

– Elastase – acts on peptide bonds of amino acid with small R group (Ala, Ser)

• Have a substrate-binding pocket that the enzyme use to identify the residue for which it is specific

ASPARTIC PROTEASE• Includes the digestive enzyme pepsin, the

lysosomal cathepsin and the protease produced by the human immunodeficiency virus (HIV)

• Catalysis involves two conserved aspartyl residues which act as acid-base catalysts

FRUCTOSE 2,6-BISPHOSPHATASE• Enzyme involved in gluconeogenesis • Catalyzes the hydrolytic release of phosphate

from carbon of fructose 2,6 bisphosphate METAL IONS• Are present in 25% of all enzymes• Classes of metal ion-requiring enzymes

1. Metalloenzymes - Contain tightly bound metal

ions, most commonly transition metal ions such as Fe++, Fe+++, Cu++, Mn++, Co++, Zn+

+

2. Metal-activated enzymes- Loosely binds metal ions from

solutions, usually the alkali and alkali earth metal ions Na+, K+, Mg++, Ca++

TYPES OF TERNARY COMPLEXES

ROLE OF METAL IONS• Serve as 3-dimensional template for

orientation of basic groups on enzyme or substrate

• Accept electrons via sigma or pi bonds to activate electrophile or nucleophile

• Mask a nucleophile and thus prevent an otherwise likely side reaction

• Coordination sphere of metal may bring together enzyme and substrate or form chelate producing distortion in either the enzyme or substrate

CARBONIC ANHYDRASE

SUMMARYTRUE OR FALSE?• Coenzymes are small, heat-stable molecules

containing a nucleotide grouping that are often involved in enzyme-catalyzed transfer reactions.

TRUE

• Enzymes that contain prosthetic groups can often be separated into a protein part, the holoenzyme, which is inactive, and a non-protein part

False. holoenzyme itself is a whole enzyme and it is active.

• Enzymes may have one or more active centers where their substrates bind and undergo chemical change

TRUE• Phosphatases are enzymes that synthesize

phosphate esters False. They remove phosphate.

• Liver and heart lactate dehydrogenase are identical

False. H (heart) and M (liver) subunits.• Isoenzymes are distinct molecular forms of

enzymes catalyzing the same reaction TRUE

• The addition of a competitive inhibitor to an enzyme reaction will increase the apparent Km for substrate

TRUE• The value of Vmax determined for an enzyme

is proportional to the amount of enzyme used when measuring values of the initial rate v

False. the initial velocity belongs to the 1st order kinetics.