chapter 3 enzymes

7/30/2019 Chapter 3 Enzymes

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Chapter 3 Enzymes


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Almost all processes in the living cell are catalyzed

by the specific biocatalyst. Enzymes are catalysts

that change the rate of a reaction without beingchanged themselves. Enzymes are highly specific and

their activity can be regulated..


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Biocatalyst: enzymes and ribozyme.

One of the most important functions of proteins is

their role as catalysts. Until recently, all enzymes were

considered to be proteins. Several examples of

catalytic RNA molecules have now been vertified.

Living processes consist almost entirely of biochemicalreactions. Without catalysts these reactions would not

occur fast enough to sustain life.


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Enzymes bind to one or more ligands,

called substratee, and convert them intoone or more chemically modified products.


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1 Composition of enzymes

Simple enzyme and conjugated enzyme.

Conjugated enzyme:

apoenzyme + cofactor holoenzyme.

Cofactor : prosthetic group+ coenzyme

prosthetic group: tightly bond with apoenzyme.

FAD, metal, etc. coenzyme loosely bond with apoenzyme. NAD,

NADP, etc.


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Active site: Each type of enzyme molecule

contains a unique, intricately shapedbinding surface called an active site.

Catalytic residues are highly conserved.

Certain amino acids, notably cysteine and

hydroxylic, acidic, orbasic amino acids,

perform key roles in catalysis.

Essential group in active site:binding

group +catalytic group. Cofactors always be

a part of the active site.


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Active site

The active site is the region of the enzyme thatbinds the substrate, to form an enzyme-substrate

complex, and transforms it into product. Theactive site is a three-dimensional entity, often a

cleft or crevice on the surface of the protein, in

which the substrate is bound by multiple weak

interactions. Two models have been proposed toexplain how an enzyme binds its substrate: the

lock-andkey model and the induced-fit model.


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2 Characteristics and mechanisms

of enzymatic reactions Characteristics

Enzymes have several remarkable properties. First,

the rates of enzymatically catalyzed reactions are

often phenomenally high. (Rate increases by factors of106or greater are common.) . Second, in marked

contrast to inorganic catalysts, the enzymes are highly

specific to the reactions they catalyze. Side products

are rarely formed. Finally, because of their complexstructures, enzymes can be regulated. This is an

especially important consideration in living organisms,

which must conserve energy and raw materials.


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Specificity: Absolute specificity, relative specificity, and

stereospecificity.

Activation energy: To proceed at a viable rate, most

chemical reactions require an initial input of energy. In

the laboratory this energy is usually supplied as heat. At

temperatures above absolute zero (-273.1C), all

molecules possess vibrational energy, which increases asmolecules are heated. Consider the following reaction:

A+B C

As the temperature rises, vibrating molecules (A and B)are more likely to collide, A chemical reaction occurs

when the colliding molecules possess a minimum amount

of energy called the activation energy.


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Uncatalyzed

activation energy

Enzymatic

activation energy

Energy

Progress of reaction

Total energyChanges of reaction

Non-enzymatic

activation energy

Substrate

Product

Activation energy


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Not all collisions result in chemical reactions

because only a fraction of the molecules have

sufficient energy.

Induced-fit hypothesis and transition state.

Substrates induce conformational changes in

enzymes. During any chemical reaction reactantswith sufficient energy will attain transition state

(a strained intermediate form) when the substrate

binds to the enzyme (inducing).


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Induced-fit Theory

substrate

enzyme

Complex of substrate-enzyme


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Mechanisms

Proximity effect and orientation arrange: For a

biochemical reaction to occur, the substrate must

come into close proximity to catalytic functional

groups (side chain groups involved in a catalytic

mechanism ) within the active site. In addition, thesubstrate must be precisely, spatially oriented to

the catalytic groups. Once the substrate is

correctly positioned, a change in the enzymes

conformation may result in a strained enzyme-substrate complex. This strain helps to bring the

enzyme-substrate complex into the transition state.


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Multielement catalysis (Acid-Base catalysis ) :

Chemical groups can often be made more reactive

by adding or removing a proton. Enzyme activesites contain side chain groups that act as proton

donors or acceptors. These groups are referred to

as general acids or general bases.

Surface effect: The strength of electrostatic

interactions is related to the capacity of

surrounding solvent molecules to reduce the

attractive forces between chemical groups. Wateris largely excluded from the active site as the

substrate binds.


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3 Enzyme kinetics

The rate orvelocity of a biochemical reaction is

defined as the change in the concentration of a

reactant or product per unit time.

Plotting initial velocity v versus substrateconcentration [S].The rate of the reaction is directly

proportional (first order reaction) to substrate

concentration only when [S] is low. When [S]

becomes sufficiently high that the enzyme is

saturated, the rate of the reaction is zero-order with

respect to substrate.


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V


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Michaelis-Menten Equation

K1= rate constant for ES formation

K2= rate constant for ES dissociation

K3= rate constant for product formation

and release from the active site

(1)S + E ES E + P

k1

k2

k3


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(2)v=V

max

[S]

Km + [S]


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ES formation = K1 ( [E] - [ES] ) [S] (3)

ES dissociation = K2 [ES ]+ K3 [ES] (4)

S + E ES E + P

k1

k2

k3


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K1 ( [E] - [ES] ) [S] = K2 [ES ]+ K3 [ES]

( [E] - [ES] ) [S] K2+ K3

=

[ES] K1


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Michaelis and Menton introduced a new constant,

Km ( now referred as the Michaelis constant):

K2+ K3

Km=K1

( [E] - [ES] ) [S]

Km=

[ ES]


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Km [ES] = [E] [S] [ES] [S]

Km [ES] + [ES] [S] = [E] [S]

[ES] ( Km + [S] ) = [E] [S]

[E] [S]

[ES] = (5)

Km+[S]


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Since V= K3 [ES], from ( 5 )

[E] [S]

V= K3 (6)

Km+[S]

When the [S] is much higher than the enzymes, all

enzymes form [ES], that is, [E]= [ES], and maximum

velocity ( Vmax

) can attain.

Vmax = K3 [ES] = K3 [E] (7)

Vmax

K3 =

[E]


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Vmax [E] [S] Vmax [S]

V= = (2)

[E] Km+[S] Km+[S]


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Significances of Km and Vmax

1) When [S] = Km,

Vmax [S] Vmax

V = =

[S] + [S] 2

2) When [S] is very much greater than Km,

Vmax [S] Vmax [S]V= = = V

max

Km+[S] [S]


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3) It may reflect the affinity of the enzyme for itssubstrate. If K3 is much smaller than K2, that is K3 K2,

Km is the dissociation constant for the [ES].K

2

Km=

K1

4) From Vmax = K3 [ES] = K3 [E], enzymes are saturated.

Vmax

K3=

[E]

The turnover number (Kcat

) = K3. This quantity is the

number of moles of substrate converted to product

each second per mole of enzyme.


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Vmaxv

1

=

Km . 1[S] + Vmax

1

Lineweaver-Burk Double-reciprocal

plot

y = mx + b


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Slope

Vmaxv

1

=

Km

.1

[S] + Vmax

1

(intercept on the vertical axis)

(intercept on the horizontal axis)


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Multiple factors affect the rates of

enzyme-catalyzed reactions.

Temperature

While raising temperature increases the rate of an

enzyme-catalyzed reaction, this holds only over a

strictly limited range of temperatures. The reactionrate initially increases as temperature rises owing to

increased kinetic energy of the reacting molecules.

Eventually, however, the kinetic energy of the enzyme

exceeds the energy barrier for breaking the weak bonds

that maintain its secondary-tertiary structure. At this

temperature, denaturation, with an accompanying

precipitate loss of catalytic activity, predominates.


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Enzymes from humans, who maintain a body

temperature of 37 C, generally exhibit stability at

temperature up to 45-55 C. Enzymes frommicroorganisms that inhabit natural hot springs or

hyperthermal vents on the ocean floor may be

stable at or above 100 C.

Optimum temperature: Temperature at which it

operates at maximal efficiency.


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Enzymeactivity

Temperature(C )


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pH

When enzyme activity is measured at several pH

values, optimal activity typically is observed betweenpH values of 5 and 9. However, a few enzymes are

active at pH values well outside this range.

pH optimum:The pH value at which an enzymes

activity is maximal is called the pH optimum.


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Initial rate is proportionate to enzyme

concentration

The initial rate of a reaction is the rate

measured before sufficient product has been

formed to permit the reverse reaction to occur.

The initial rate of an enzyme-catalyzed reaction isalways proportionate to the concentration of

enzyme. Note, however, that this is statement

holds only for initial rates.

Substrate concentration


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[S]>>[E]

[E]v


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pH dependent of enzyme activities

Pepsin AmylaseAcetylcholinesterase

Enzymeactivity

pH


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(4) Enzyme inhibition

The activity of enzymes can be inhibited. Many substances

can reduce or eliminate the catalytic activity of specific

enzymes. Inhibition may be irreversible orreversible.

Irreversible inhibitors usually bond covalently to the

enzyme, often to a side chain group in the active site. For

example, enzymes containing free sulfhydryl groups can

react with alkylating agents such as iodoacetate and heavy

metals. This process is not readily reversed either by

removing the remainder of the free inhibitor or by

increasing substrate concentration.

Specific inhibitor: specifically bind to essential amino acid

on active site. Some organic phosphor compounds could

specifically bind toOH of serine.


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Non specific inhibitor: not only binds to essential

group, but also to outsides of essential group. Hg2+,

Ag2+ and As3+ .

In reversible inhibition:

the inhibitor can dissociate from the enzyme because it

binds through noncovalent bonds. The most common

forms of reversible inhibition are competitive and

noncompetitive.


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1) Competitive inhibition

Competitive inhibitors typically resemblethe substrate

Classic competitive inhibition occurs at

the substrate-binding (catalytic) site. Thechemical structure of a substrate analog

inhibitor (I) generally resembles that of the

substrate (S). It therefore combinesreversibly with the enzyme, forming an

enzyme-inhibitor (EnzI) complex rather than

an EnzS complex.


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Competitive inhibition


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Vmaxv1

=Km 1

[S]+

Vmax

1(1+

Ki

[I]))

v=

Vmax [S]

Km (1+ + [S]Ki

[I]))

E + S E + P

+I

EI

ES

Ki


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inhibitor

No inhibitor


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Noncompetitive inhibition

In noncompetitive inhibition, no competition

occurs between S and I. The inhibitor

usually bears little or no structural

resemblance to S and may be assumed tobind to the enzyme at a site other than the

active site. Both EI and EIS complexes

form. Inhibitor binding alters the enzymesthree-dimensional configuration and blocks

the reaction.


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Noncompetitive inhibition


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E + S ES E + P+

I

ESI

+

I

EI + S

E + S ES E + P+

I

ESI

+

I

EI + S

Ki Ki


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Plots of 1/V versus 1/[S] in the

presence of several concentrations of theinhibitor intersect at the same point on

the horizontal axis, -1/Km. In

noncompetitive inhibition the dissociationconstants for ES and EIS are assumed to

stay the same.


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inhibitor

No inhibitor


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3) Uncompetitive inhibition

The inhibitor bind to ES and results indecrease of both ES and P (also free E).

E + S ES E+S

+

I

Ki

ESI


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Uncompetitive inhibition


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Ki

E + S ES E + P

ESI

+

I


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)K

I

(1V

1

S

1

V

K

v

1

imaxmax

m


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No inhibitor

inhibitor


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4) Effect of activator on the enzyme

activities Activator: substances enable non-active

enzyme to become active one. Metals such

as Mg2+, K+, Mn2+, etc. Essential activator and non-essential

activator.


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5) Enzyme activity assay and unit of

enzyme activity Enzyme activity is measured in international units

(I.U.) One I.U. is defined as the amount of enzyme

that produces 1mol of product per minute. An

enzyme specific activity, a quantity that is used tomonitor enzyme purification, is defined as the number

of international units per milligram of protein.

A new unit for measuring enzyme activity called the

katal,has recently been introduced. One katal (kat)indicates the amount of enzyme for the transformation

of 1 mole of substrate per second.

1 IU =16.67

10-9

kat


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4 Regulation of enzyme

The thousands of enzyme-catalyzed chemicalreactions in living cells are organized into a

series of biochemical or metabolic pathways.

Each pathway consists of a sequence ofcatalytic steps. The product of the first

reaction becomes the substrate of the next

and so on. Metabolic and other processes arecontrolled by altering the quantity or the

catalytic efficiency of enzymes.


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1) Regulation of enzyme activities

A. Proenyme orZymogen: Certain proteins are

manufactured and secred in the form ofinactive

precursor proteins known as proproteins. When the

proteins are enzymes, the proproteins are termed

proenzymes or zymogens. Conversion of a

proprotein to the mature protein involves selective

proteolysis, a process that converts the proprotein by

one or more successive proteolytic clips to a form

having the characteristic activity of the matureprotein ( its enzymatic activity ). Examples include

the hormone insulin (proinsulin), pepsinogen,

trypsinogen, etc.


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Selective proteolysis of a proenzyme may

be viewed as a process that triggers

essential conformational changes that

create the catalytic site.


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B. Allosteric enzyme

Allosteric enzymes are enzymes whoseactivity at the catalytic site may be modulated by

the presence of allosteric effectors at an allostericsite. Allosteric effector could be products,

substrate, and so on.

Feed back inhibition referred to the inhibition of

the activity of an enzyme in a biosyntheticpathway by an end product (often as allosteric

effectors) of that pathway.


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C. Regulatory covalent modification

Regulatory covalent modifications can be

reversible or irreversible. In mammalian cells,

the two most commonly used forms of covalent

modification are partial proteolysis andphosphorylation. Because cells lack the ability

to reunite the two portions of a protein

produced following hydrolysis of a peptidebond, the partial proteolysis is considered an

irreversible modification.


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Hydrolysis of the phosphoesters formed

when a protein is covalently phosphorylated

on the side chain of a serine, threonine, or

tyrosine residues is both thermodynamically

spontaneous and readily catalyzed by

enzymes called protein phosphatases. Hence,phosphorylation represents a reversible

modification process.


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Cyclic phosphorylation

and dephosphorylation

is a common cellularmechanism for

regulating protein

activity. In this example,

the target protein R(orange) is inactive when

phosphorylated and

active when

dephosphorylated; the

opposite pattern occurs insome proteins.


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2) Regulation of enzyme quantity

Rate of synthesis and degradation determine enzymequantity. The quantity of an enzyme in a cell may be

increased either by elevating its rate of synthesis, by

decreasing its rate of degradation, or by both. Cells can

synthesize specific enzymes in response to changingmetabolic needs, a process referred to as enzyme

induction. The induction accomplished by genetic

control. Although many inducers are substrates for the

enzymes they induce, compounds structurally similar tothe substrate may be inducers but not substrates.

Conversely, a compound may be a substrate but not an

inducer.


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The synthesis of certain enzymes may also be

specifically inhibited. In a process called repression,

the end product of a biochemical pathway mayinhibit the synthesis of a key enzyme in the pathway.

Both induction and repression involve cis-elements,

specific DNA sequences located upstream of genes

that encode a given enzyme, and a trans-actingregulatory proteins.

Regulation of enzyme degradation. The

degradation of mammalian proteins by ATP andubiqitin-dependent pathways and by ATP-

independent pathways. It also Related to the

nutrition and hormone state.

C i


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Compartmentation

In eukaryotic cells, biochemical pathways aresegregated into different organelles. One purpose forthis physical separation is that opposing processes are

easier to control if the occur in different

compartments. For example, fatty acid biosynthesisoccurs in the cytoplasm, while the energy-generating

reactions of fatty acid oxidation occur within the

mitochondria. Another factor is that each organelle

can concentrate specific substances such as substratesand coenzymes. In addition, special

microenvironments are often created within

organelles.


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3) Isoenzymes

The enzymes catalyzing the same

biochemical reaction.

Lactate dehydrogenase (LDH)


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Isoenzymes

H subunit M subunit

Isoenzymes of lactate dehydrogenase


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5 Nomenclature and classification

The International Union of Biochemistry

(IUB) adopted a complex but unambiguous

system of enzyme nomenclature based onreaction mechanism.

(1) Reactions and the enzymes that

catalyzed them form six classes, eachhaving 4-13 subclasses.


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(2) The enzyme name has two parts. The first

names the substrate or substrates. The second,

ending inase, indicates the type of reactioncatalyzed.

(3) Additional information, if needed to clarify the

reaction, may follow in parentheses; eg, the enzyme

catalyzing

L-malate + NAD+ pyruvate + CO2 + NADH + H+

is designated 1.1.1.37 L-malate:

NAD+ oxidaoreductase (decarboxylating). (4) Each enzyme has a code number (EC) that

characterizes the reaction type as to class, subclass,

and subsubclass.


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Classification

Six classes based on reaction mechanism:

(1) Oxidoreductases: LDH, Cytochrome C, etc.

(2) Transferases: methyl transferase.

(3) Hydrolases: amylase (4) Lyases removing a group to form a double bond,

or reverse reaction.

(5) Isomerase to catalyze the intertransfer of isomers. (6) Ligase. catalyzing two substrates link to form one

compound.


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A

B

C

D

E

1.


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A

B

C

D

E

2


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3. ( )A.

B. C.

D.

E.


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4. Holoenzyme refer to ( )

A. Complex of enzyme with substrate

B. Complex of enzyme with suppressant

C. Complex of enzyme with cofactor

D. Inactive precursor of enzyme

E. Complex of enzyme with allosteric effector


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5. ( )A.

B.

C.

D.

E.


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6. ( )A.

B.

C.

D.

E.


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8. Michaelis-Menten enzyme kineticsdiagram of curves is a ( )

A. straight line

B. rectangular hyperbola

C. S shape curve

D. parabola

E. Not above all


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10. ( )A.

B.

C.,

D. ,

E.


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12. ( )A.

B. -C.

D.

E. -


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13. ( )A.

B. ,

C.

D. ,

E. ,


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14. SH is one enzymes essential group. Whichsubstance can protect this enzyme from oxidation?

A. Cys

B. GSH

C. urea

D. ionic detergent

E. ethanol


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15. ( )A.

B.

C.

D.

E.


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16. The characteristic constants ofenzymes include ( )

A. Enzymic optimum temperature

B. Enzymic optimum pH

C. Vmax

D. Km

E. KS


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17. ( )A.

B.

C.

D.

E.


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18. Cofactors of enzyme are ( )

A. Micromolecule organic compounds

B. metal ion

C. vitamine

D. various kinds of organic and

inorganic compoundsE. A kind of conjugated protein


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19. -SH,( )A. GSH

B.C

C.

D.A

E.


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20. ( )

A.

B.

C.

D.

E.


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Thank you!

chapter 3 enzymes

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