C1 Introduction to enzymes

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C Enzymes

• C1 Introduction to enzymes
• C2 Thermodynamics???
• C3 Enzyme kinetics???
• C4 Enzyme inhibition
• C5 Regulation of enzyme activity

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C1 Introduction to enzymes

• Enzyme as catalysts???
• Active site ????
• Substrate specificity?????
• Enzyme classification??
• Enzyme assays????
• Linked enzyme assays
• Coenzymes?? and prosthetic groups??
• Isoenzymes???

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Enzymes as catalysts

• Enzymes are catalysts that change the rate of a
  reaction without being changed themselves
• Enzyme catalyzed reactions usually take place
  under relatively mild conditions(T,P,pH).
• Enzymes are highly specific and their activity
  can be regulated.
• Virtually all enzymes are proteins, although some
  catalytically active RNAs have been identified.
•  

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Active site????

• Active site is the region that binds the
  substrate ??and converts it into product?
• ????????????,????????
• ??????????????????,?????????
• Small(1-2)?a three-dimensional entity??
• Cleft or crevice??on the surface of enzyme
• Bind by multiple weak forces
• Enzyme-substrate complex
• transition state complex??????--?product

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Active site
Active site



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Enzyme-substrate complex
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Two models---enzyme bind its substrate

• Lock and key model?-?Emil Fischer,1894
• Induced fit model????Daniel E. Koshland,Jr 1958

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Lock and key model
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Induced fit model
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Induced fit model

• Active sites in the uninduced enzyme are shown
  schematically with round contours. Binding of the
  first substrate (gold) induces a conformational
  shift (angular contours) that facilitates binding
  of the second substrate (blue), with far lower
  energy than otherwise required. When catalysis is
  complete, the product is released, and the enzyme
  returns to its uninduced state.

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Substrate specificity

• Substrate specificity is often determined by
  changes in relatively few amino acids in the
  active site.
• ?????
• ?????(??)
• ?????(?????????????)
• ???????
• ???????(L-??????)
• ???????

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Serine proteases????????

• Trypsin???? cleaves on the carboxyl side of
  positively charged Lys or Arg residues.
• Chymotrypsin ?????? cleaves on the carboxyl side
  of bulky aromatic and hydrophobic amino acid
  residues
• Elastase ????? cleaves on the carboxyl side of
  small uncharged side chains

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Enzyme classification
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2. Enzyme classification
(1) ??-??? Oxidoreductase

• ??-???????-?????
• ???????(dehydrogenase)????(Oxidase)?
• ?,??(Lactate)?????????????

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2. Enzyme classification
(2) ??? Transferase

• ???????????,?????????????????????????????,
  ???????????????

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2. Enzyme classification
(3) ??? hydrolase

• ???????????????
• ????????????????????
• ??,???(Lipase)?????????

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Enzyme classification
(4) ??? Lyase

• ?????????????????????????????????
• ?????????????????
• ??, ?????????????

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Enzyme classification
(5) ??? Isomerase

• ?????????????????,???????????????????,6-?????????
  ?????

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Enzyme classification
(6) ??? Ligase or Synthetase

• ???,??????,????C-C?C-O?C-N ??C-S
  ??????????????ATP?????????
• A B ATP H-O-H A ? B ADP Pi
• ??,????????????
• ??? CO2 ? ????

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Enzyme classification
(7) ???(????) ribozyme

• ???????????????????RNA,????RNA?????????????????

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Enzyme classification

• Trypsins Enzyme Commission number is 3.4.21.4
• 3 denote it is a hydrolase
• 4 denote it is a protease that hydrolyzes peptide
  bonds
• 21 denote it is a serine protease with a serine
  residue at the active site
• 4 indicates that it was the fourth enzyme to be
  assigned to this class.

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Enzyme assays

• The amount of enzyme protein present can be
  determined in terms of the catalytic effect it
  produces,that is the conversion of substrate to
  product.
• The overall equation of the reaction
• The disappearance of substrate or the appearance
  of product
• Cofactors,pH ,temperature
• Sufficient supply of substrate

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Enzyme assay

• Enzyme activity(????????????)the rate of
  appearance of product or the rate of
  disappearance of substrate
• Absorbancespectrophotometer?????
• Two most common molecules
• NADH(reduced nicotinamide ademine
  dinucleotied?????????????)
• NADPH340nm

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NADH (NADPH)
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LDHLactate dehydrogenase
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Linked enzyme assays

• If neither the substrates nor products of an
  enzyme-catalyzed reaction absorb light at an
  appropriate wavelength,the enzyme can be assayed
  by linking to another enzyme-catalyzed reaction
  that does involve a change in absorbance.
• The second enzyme must be in excess,so that the
  rate-limit step in the linked assay is the action
  of the first enzyme.

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Coenzymes and prosthetic groups

• Cofactorssmall,nonprotein units(iorganic ions or
  a complex organic molecule called coenzyme??)
• Prosthetic group ??A metal or coenzyme that is
  covalently attached to the enzyme is called a
  prosthetic group.
• Holoenzyme??cofactor???apoenzyme???
• Many coenzymes are derived from vitamin
  precursors,giving rise to deficiency diseases
  when in inadequate supply.
• P73 table 2

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Cofactors
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NAD(??I) and NADP(??II)

• NAD and NADP have a common function as they
  both act as carriers of electrons and are
  involved in oxydation-reduction reactions.
• NAD is more commonly used in catabolic(break
  down)????reactions
• NADP is used in anabolic(biosynthetic)????reacti
  ons.
• The reactive part of both molecules is the
  nicotinamide ring which exists in reduced or an
  oxidized form, and so acts to accept or donate
  electrons in an enzyme reaction.
• NAD H2e- lt ----- gt NADH

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NAD
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NADP
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FAD and FMN

• Flavin adenine dinucleotide(FAD) ??????? ??and
  flavin mononucleotide(FMN) ??????are also
  carriers of electrons .
• Reactive site is flavine mononucleotide unit.
• FAD and FMN react with two protons as well as two
  electrons,in alternating between the reduced and
  oxidized state.
• FAD2H2e-lt ----- gtFADH2

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FMN
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FAD
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Isoenzymes???

• Isoenzymes are different forms of an enzyme which
  catalyze the same reaction,but which exhibit
  different physical or kinetic properties(pI,pH,
  substrate affinity or inhibitors)
• LDH?????
• CH3CH(OH)COO-NADlt ----gtCH3COCOO-NADHH

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LDH

• LDH is a tetramer of two different types of
  subunits,called H and M,which have small
  differences in amino acid sequence.
• The two subunits can combine randomly with each
  other,forming 5 isoenzymes that have the
  compositions H4, H3M,H2M2,HM3,M4.
• They can be resolved electrophoretically.
• M subnunits predominate in skeletal muscle and
  liver,whereas H subunits predominate in the
  heart.
• H4, H3Mheart and red blood cells
• H2M2brain and kidney
• HM3,M4 liver and skeletal muscle

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C2 Thermodynamics

1. Thermodynamics???
2. Activation energy ???and transition state
3. Free energy change
4. Chemical equilibria????

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Thermodynamics

• A knowledge of thermodynamics, (which is the
  description among the various forms of energy and
  how energy affects matter),enables one to
  determine whether a physical process is possible.
  
• In Thermodynamics, a system is the matter within
  a defined region.
• The matter in the rest of the universe is called
  the surroundings.

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The first laws of thermodynamics

• ?EEA-EBQ-W
• EA is the energy of the system at the start of a
  process
• EB is the energy of the system at the end of the
  process
• Q is the heat absorbed by the system
• W is the work done by the system.
• Q ?E W ????????????????????????????????????????
  ?

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The second laws of thermodynamics

• The second laws of thermodynamics states that a
  process can occur spontaneously only if the sum
  of the entropies ?(Entropy is a measure of the
  degree of randomness or disorder of a system)of
  the system and its surroundings increases.
• ?S system ? S surroundings gt0 for a spontaneous
  process
• Entropy changes of chemical reactions are not
  readily measured.

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Free energy???(G)

• Josiah Willard Gibbs
• The first and second laws of thermodynamics are
  combined in the thermodynamic function, free
  energy.
• ?G ?H-T ?S(?H??????S??)
• ?H ?EP ?V
• ?G ?E-T ?S
• If ? G is negative, that reaction can happen
  spontaneously
• If ? G is positive,an input of energy is
  required to drive the reaction.

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Activation energy and transition state

• For a biochemical reaction to proceed, the
  energy barrier needed to transform the substrate
  molecules into the transition state has to
  overcome.
• The transition state has the highest free energy
  in the energy pathway.

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Gibbs ?????

• ?G(Gibbs ?????)The difference in free energy
  between the substrate and transition state is
  termed the Gibbs free energy of activation.

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?G

• An enzyme stabilizes the transition state and
• lowers ?G ,thus increasing the rate at which
  the reaction occurs.

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?G and ?G
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?Go

• ATPH2O-------gtADPPi
• ?Go-30.5kJ mol-1-7.3kcalmol-1
• 1kcal?4.184kJ

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Chemical equilibria

• A chemical reaction often exists in a state of
  dynamic equilibrium.
• The equilibrium constant ( K) defines the ratio
  of the substrates and product at equilibrium.
• Enzymes do not alter the equilibrium position,but
  do accelerate the attainment of the equilibrium
  position by speeding up the forward and reverse
  reactions.

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C3 Enzyme kinetics

• Enzyme velocity
• Substrate Concentration
• Enzyme concentration
• Temperature
• pH
• Michaelis-menten Model
• Lineweaver-Burk Plot

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Enzyme velocity

• Enzyme activity is commonly expressed by the
  intial rate ( V0) of the reaction being
  catalyzed. The units of V 0are u mol/ min? (why?)
  

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Enzyme unit

• Enzyme activity may be expressed in a number of
  ways, the commonest is by the V0
• There are also 2 standard units of enzyme
  activity
• the enzyme unit (U) and the katal(kat),
• 1Uthe amount of enzyme which will catalyze the
  transformation of 1 u mol of substrate per min at
  25oC under optimal conditions .
• 1kat??????,??????1mol?????????????
• 1U16.67 nkat.

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Specific activity???

• The specific activity is the number of units per
  milligram of protein(units /mg)
• Or ?g?????ml??????????????(U/g,U/ml)??????????????
  ?????
• The specific activity is a measure of the purity
  of an enzyme.

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Substrate Concentration

• At low substrate concentrations, a doubling of
  S leads to a doubling of V ? ,whereas at higher
  S the enzyme becomes saturated and there is no
  further increase in V ?

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Substrate Concentration

1. ???????
2. ????
3. ???

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Enzyme concentration

• When S is saturating,a doubling of the enzyme
  concentration leads to a doubling of V0.

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Temperature

• Temperature affects the rate of an
  enzyme-catalyzed reaction by increasing the
  thermal energy of the substrate molecules.
• This increases the proportion of molecules with
  sufficient energy to overcome the activation
  barrier and hence increases the rate of the
  reaction .
• in addition ,the thermal energy of the component
  molecules of the enzyme is increased,which leads
  to an increased rate of denaturation of the
  enzyme protein due to the disruption of the
  noncovalent interactions holding the structure
  together.

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T
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pH

• Each enzyme has an optimum pH at which the rate
  of the reaction that it catalyzes is at its
  maximum.
• Slight deviations in the pH from the optimum lead
  to a decrease in the reaction rate .
• Larger deviations in pH lead to denaturation of
  the enzyme due to charges in the ionization of
  amino acid residues and the disruption of
  noncovalent interactions

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pH
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Michaelis-menten Model

• Vmax???????????,S?????,Km?????,VO???????????????
  ??

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Michaelis-Menten model
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Michaelis-Menten model
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Michaelis-Menten model
k1
ES-------gtES
K-1
ES-----gt ES
k2
ES------- gtEP
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Michaelis-Menten model
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Michaelis-Menten model

• The concentration of uncombined enzyme E is
  equal
• to the total enzyme concentration ET minus the
  concentration of the EScomplex.

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Michaelis-Menten model
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Michaelis-Menten model

• The maximal rate, Vmax, is attained when the
  catalytic sites on the enzyme are saturated with
  substratethat is, when ES ET. Thus,

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A direct plot
??????????????, ?V 1/2 Vmax, Km S
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Km
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Kcat????,????????????????????
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A lineweaver-burk double-reciprocal plot
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C4 Enzyme inhibition

1. Enzyme inhibition
2. Irreversible inhibition
3. Reversible competitive inhibition
4. Reversible noncompetitive inhibition

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Enzyme inhibition

• Inhibitorany molecule which acts directly on an
  enzyme to lower its catalytic rate is called an
  inhibitor.(not denaturation)
• Some enzyme inhibitors are normal body
  metabolites,
• other may be foreign substances,such as drugs or
  toxins

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Irreversible

• IrreversibleAn irreversible inhibitor binds
  tightly, often covalently, to amino acid residues
  at the active site of the enzyme, permanently
  inactivating the enzyme.
• ?????????
• ????????

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diisopropylfluorophosphate( DIPF)
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Iodoacetamide ????
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Antibiotic penicillin
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Penicillin
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Irreversible
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e.g
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Reversible Competitive inhibition

• A competitive inhibitor typically has colse
  structural similarities to the normal substrate
  for the enzyme.
• Thus it competes with the substrate molecules for
  binding to the active site of the enzyme.
•  

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Reversible Competitive inhibition

• At high substrate concentration, the effect of
  a competitive inhibitor can be overcome.

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Succinate dehydrogenase
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Reversible Competitive inhibition

• On a Lineweaver-Buck plot a competitive can be
  seen to increase the Km but leave Vmax unchanged

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Reversible competitive inhibition



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Reversible noncompetitive inhibition

• A noncompetitive inhibitor binds at a site
  other than the active site of the enzyme and
  decreases its catalytic rate by causing a
  conformational change in the three-dimen-sional
  shape of the enzyme.

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Reversible noncompetitive inhibition

• The enzyme may bind the inhibitor,the substrate
  or both the inhibitor and substrate together.

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Reversible noncompetitive inhibition

• The effect of a noncompetitive inhibitor cannot
  be overcome at high substrate concentrations.
• On a Lineweaver-Buck plot a noncompetitive
  inhibitor can be seen to decrease the Vmax but
  leave Km unchanged.

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Lineweaver-Buck plot
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Competitive inhibition and noncompetitive
inhibition
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Competitive inhibition and noncompetitive
inhibition
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C5 regulation of enzyme activity

1. Feedback regulation????
2. Allosteric enzyme???
3. Reversible covalent modification
4. Proteolytic activation???????
5. Regulation of enzyme synthesis and breakdown

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Feedback regulation????

• The rate of enzyme-catalyzed reactions in
  biological systems are altered by activators and
  inhibitors,
• (collectively known as effectors.)
• In metabolic pathways, the end-product often
  feedback-inhibits (when an enzyme early on in the
  pathway is inhibited by an end-product of the
  metabolic pathway)the committed step earlier in
  the same pathway to prevent the build up of
  intermediates and the unnecessary use of
  metabolites and energy.

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Feedback inhition loop
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Sequential feedback inhibition

• For branched metabolic pathways a process of
  sequential feedback inhibition often operates.

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Allosteric enzyme

• A plot of V 0 against S for an allosteric
  enzyme gives a sigmoidal-shaped curve.

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Allosteric enzyme

• Allosteric enzymes are often multi-subunit
  proteins,with an active site on each subunit.
• They often have more than one active site which
  co-operatively bind substrate molecules,such that
  the binding of substrate at one active site
  induces a conformational change in the enzyme
  that alters the affinity of the other active
  sites for substrates.
• In addition,allosteric enzymes may be controlled
  by effectors (activators or inhibitors) that bind
  to a site other than the active site and alter
  the rate of enzyme activity.

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Allosteric enzyme
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Aspartate transcarbamonylase
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ATCase ?????????

• This enzyme consist of six catalytic subunits
  each with an active site and six regulatory
  subunits to which the allosteric effectors
  cytosine triphosphate (CTP) and ATP bind.

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Allosteric activator

• Aspartate transcarbamoylase is feedback-inhibitor.
  
• In contrast,ATP an intermediate earlier in the
  pahway,acts as an allosteric activator.

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Allosteric
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Reversible covalent modification

• The activity of many enzymes is alterd by the
  reversible making and breaking of a covalent bond
  between the enzyme and a small nonprotein group.

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Covalent modifation
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Phosphorylation and dephosphorylation

• The most common such modification is the addition
  and removal of a phosphate groupphosphorylation
  and dephosphorylation????,respectively.
  Phosphorylation is catalyzed by protein
  kinases,????often using ATP as the phosphate
  donor,
• whereas dephosphorylation is catalyzed by
  protein phosphatases???????

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Phosphorylation
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Phosphorylation and dephosphorylation
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Proteolytic activation

• Some enzymes are synthesized as larger inactive
  precursors calles proenzymes or zymogens??.
• These are activated by the irreversible
  hydrolysis of one or more pepide bonds.
• The pancreatic proteases trypsin,chymotrypsin and
  elastase are all derived from zymogen precursors
  by proteolytic activation.
• Premature activation of these zymogens leads to
  the condition of acute pancreatitis?????.

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The central role of trypsin
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Activation of chymotrypsinogen
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The blood clotting cascade

• The blood clotting cascade also involves a series
  of zymogen activations that brings about a large
  amplification of the original signal.

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Clotting cascade
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Clotting cascade
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Regulation of enzyme synthesis and
breakdown

• The amount of enzyme present is a balance between
  the rates of its synthesis and degradation,The
  level of induction or repression of the gene
  encoding the enzyme,and the rate of degradation
  of its mRNA,will alter the rate of synthesis of
  the enzyme protein.
• Once the enzyme protein has been synthesized,the
  rate of its breakdown (half-life) can also be
  altered a means of regulating enzyme activity.

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cAMP
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cAMP
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Example one
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Chymotrypsin

• (a) A representation of primary structure,
  showing disulfide bonds and the location of key
  amino acids. Note that the protein consists of
  three polypeptide chains. The active-site amino
  acids are found grouped together in the
  three-dimensional structure.
• (b) A space-filling model of chymotrypsin. The
  pocket in which the aromatic amino acid side
  chain is bound is shown in green.
• (c) The polypeptide backbone of chymotrypsin
  shown as a ribbon structure. Disulfide bonds are
  shown in yellow the A, B, and C chains are shown
  in dark blue, light blue, and white,
  respectively.

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Chymotrypsin

• (d) A close-up of the chymotrypsin active site
  with a substrate bound. Ser195 attacks the
  carbonyl group of the substrate (shown in
  purple) the developing negative charge on the
  oxygen is stabilized by the oxyanion hole (amide
  nitrogens shown in orange) In the substrate, the
  aromatic amino acid side chain and the amide
  nitrogen of the peptide bond to be cleaved are
  shown in light blue.

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Chymotrypsin