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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!
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