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 being changed 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 biochemical reactions. 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 into one 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 shaped binding
  surface called an active site.
• Catalytic residues are highly conserved. Certain
  amino acids, notably cysteine and hydroxylic,
  acidic, or basic 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 that
  binds the substrate, to form an enzyme-substrate
  complex, and transforms it into product. The
  active 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 to
  explain how an enzyme binds its substrate the
  lock-and key 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 of 106or 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 complex structures, 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 (-273.1ºC), all molecules possess
  vibrational energy, which increases as molecules
  are heated. Consider the following reaction
• AB 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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Activation energy
Uncatalyzed activation energy
Energy
Non-enzymatic activation energy
Enzymatic activation energy
Substrate
Total energy Changes of reaction
Product
Progress of reaction
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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 reactants
  with 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
Complex of substrate-enzyme
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, the substrate 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 active
  sites 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. Water
  is largely excluded from the active site as the
  substrate binds.

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3 Enzyme kinetics

• The rate or velocity 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 substrate
  concentration 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
(1)
K1 rate constant for ES formation K2 rate
constant for ES dissociation K3 rate constant
for product formation and release from
the active site
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(2)
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ES formation K1 ( E - ES ) S
(3) ES dissociation K2 ES K3 ES
(4)
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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) KmS
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Since V K3 ES, from ( 5 )
E S V K3
(6)
KmS 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 KmS KmS
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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
  Vmax
• KmS S

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• 3) It may reflect the affinity of the enzyme
  for its substrate. If K3 is much smaller than K2,
  that is K3 K2, Km is the dissociation constant
  for the ES.
• K2
• 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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Lineweaver-Burk Double-reciprocal plot
y mx b
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Slope
(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 reaction rate 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 ºC, generally exhibit stability
  at temperature up to 45-55 ºC. Enzymes from
  microorganisms that inhabit natural hot springs
  or hyperthermal vents on the ocean floor may be
  stable at or above 100 ºC.
• Optimum temperature Temperature at which it
  operates at maximal efficiency.

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Enzyme activity
Temperature ( C )
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• pH
• When enzyme activity is measured at several
  pH values, optimal activity typically is observed
  between pH 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
  is always proportionate to the concentration of
  enzyme. Note, however, that this is statement
  holds only for initial rates.
• Substrate concentration

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???????????

• ?SgtgtE?,
• E?v???
• ???

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pH dependent of enzyme activities
Enzyme activity
Acetylcholinesterase
Amylase
Pepsin
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 or reversible.
• 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 to OH
  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 resemble the
  substrate
• Classic competitive inhibition occurs at the
  substrate-binding (catalytic) site. The chemical
  structure of a substrate analog inhibitor (I)
  generally resembles that of the substrate (S). It
  therefore combines reversibly with the enzyme,
  forming an enzyme-inhibitor (EnzI) complex rather
  than an EnzS complex.

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Competitive inhibition
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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 to bind to the enzyme at a
  site other than the active site. Both EI and EIS
  complexes form. Inhibitor binding alters the
  enzymes three-dimensional configuration and
  blocks the reaction.

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Noncompetitive inhibition
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Plots of 1/V versus 1/S in the presence of
several concentrations of the inhibitor intersect
at the same point on the horizontal axis, -1/Km.
In noncompetitive inhibition the dissociation
constants 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 in decrease
  of both ES and P (also free E).
• E S ES ES
• I
• Ki
• ESI

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Uncompetitive inhibition
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inhibitor
No 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 1µmol of product per minute.
  An enzyme specific activity, a quantity that is
  used to monitor 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 16.6710-9 kat

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4 Regulation of enzyme

• The thousands of enzyme-catalyzed chemical
  reactions in living cells are organized into a
  series of biochemical or metabolic pathways. Each
  pathway consists of a sequence of catalytic
  steps. The product of the first reaction becomes
  the substrate of the next and so on. Metabolic
  and other processes are controlled by altering
  the quantity or the catalytic efficiency of
  enzymes.

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1) Regulation of enzyme activities

• A. Proenyme or Zymogen Certain proteins are
  manufactured and secred in the form of inactive
  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 mature protein (
  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 whose activity at
  the catalytic site may be modulated by the
  presence of allosteric effectors at an allosteric
  site. Allosteric effector could be products,
  substrate, and so on.
• Feed back inhibition referred to the inhibition
  of the activity of an enzyme in a biosynthetic
  pathway 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 and
  phosphorylation. Because cells lack the ability
  to reunite the two portions of a protein produced
  following hydrolysis of a peptide bond, 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 cellular mechanism for regulating protein
activity. In this example, the target protein R
(orange) is inactive when phosphorylated and
active when dephosphorylated the opposite
pattern occurs in some proteins.
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2) Regulation of enzyme quantity

• Rate of synthesis and degradation determine
  enzyme quantity. 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 changing
  metabolic 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 to the 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 may inhibit 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-acting regulatory proteins.
• Regulation of enzyme degradation. The
  degradation of mammalian proteins by ATP and
  ubiqitin-dependent pathways and by
  ATP-independent pathways. It also Related to the
  nutrition and hormone state.

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Compartmentation

• In eukaryotic cells, biochemical pathways are
  segregated into different organelles. One purpose
  for this physical separation is that opposing
  processes are easier to control if the occur in
  different compartments. For example, fatty acid
  biosynthesis occurs 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 substrates and
  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 on reaction mechanism.
• (1)    Reactions and the enzymes that catalyzed
  them form six classes, each having 4-13
  subclasses.

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• (2)    The enzyme name has two parts. The first
  names the substrate or substrates. The second,
  ending in ase, indicates the type of reaction
  catalyzed.
• (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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Relationship between Enzyme and Medicine
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????? ???
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1. ?????????????????
A.??????????? B.?????????? C.??????????????? D.??
?????????????? E.??????????????????
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2.???????????????????
A.?????????????? B.???????????????? C.??????????
?????? D.????????????? E.??????????
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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
• 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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7. ????????????( )
A. ?????? B. ?????? C. ?????? D.
???????? E. ???????????
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8. Michaelis-Menten enzyme kinetics diagram
of curves is a ( )
A. straight line B. rectangular hyperbola
C. S shape curve D. parabola E. Not above
all
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9. ??Km???????( )
A. Km?????? B. 1/Km??,????????? C.
Km????mmol/min D. Km??????????? E.
Km????????
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10. ??????????( )
A. ??????????? B. ???????????? C.
??????????,????? D. ????????,??????? E.
???????????????
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11. In anticompetitive inhibition of enzyme,
the reaction kinetics parameter change as ( )

• Km?,Vmax invariably
• Km?,Vmax?
• Km invariably,Vmax?
• D. Km?,Vmax invariably
• E. Km?,Vmax?

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12. ?????????????????( )
A. ???????? B. ?????e-?? C. ??????? D.
???????? E. ??????-??
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13. ????????????( )
A. ????????? B. ??????????,?????????????????? C.
?????????????????? D. ?????????,???????????????
??? E. ????????????,????????????????????

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14. SH is one enzymes essential group. Which
substance 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 of enzymes
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 compounds E. 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!