Phosphoryl Transfer

1
Phosphoryl Transfer

• In biological systems, the element phosphorous
  almost always exists as phosphate. Phosphorous
  is stable in several different oxidation states,
  but in phosphate, the oxidation state is 5.
  Therefore, the phosphorous atom in phosphate will
  always behave as an electrophile.
• Phosphorous can form more than four covalent
  bonds. As a second-row element, it has low lygin
  d orbitals into which additional electron pairs
  can be put to form a fifth bond. In the
  phosphate group, the unshared electron pair on
  one of the oxygen atoms can be shared with a d
  orbital of the phosphorous to form a d-pp bond.

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Examples of Phosphoryl Groups in Biochemistry
3
Small Phosphoryl-Containing Molecules
4
Phosphoryl Amino Acids
5
Classes of Phosphoryl Transfer
In kinases, X is almost always ADP. However, GDP
is known to substitute i some cases.
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Kinases

• Kinases are phosphotransferases that catalyze the
  transfer of a phosphoryl group to an acceptor
  molecule. Most often, the phosphoryl group comes
  from the terminal (gamma) position of adenosine
  triphosphate.
• There is high negative charge associated with the
  triphosphate group of ATP, which shields each
  phosphorus against reaction with incoming
  nucleophiles. This property makes ATP
  kinetically stable int he cell, although
  thermodynamically, its hydrolysis is favorable.
  In enzyme catalysis, these charges are typically
  neutralized in order to facilitate nucleophilic
  attack.
• Coordination with metal ions. Most often
  magnesium. In the cell, ATP is frequently found
  associated with magnesium, and the true substrate
  is MgATP.
• Ion pairing with positively charged amino acids
  such as the guanidinium of arginine, or the
  lysine ammonium group.
• From the structure of ATP, chemical precedent
  would indicate that the g-bond would be cleaved
  via a dissociative transition state, while the a
  and b-bonds would be cleaved via associative
  transition states.

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Adenylate Kinase

• The transfer of phosphoryl groups between
  different nucleotides, as well as other small
  molecules is important for utilizing and
  replenishing the cellular pool of energy-rich
  phosphate compounds. Adenylate kinase is a
  classic example of enzymes in this class.
• Adenylate kinase was formerly known as myokinase
  because it is found in high concentrations in
  muscle tissue.
• The adenylate kinase reaction is isoenergetic. A
  phosphoanhydride is cleaved and formed on both
  sides of the equation.
• Adenylate kinase displays sequential kinetics, in
  which both substrates must be bound before any
  product is released.
• This is distinguished from what is termed
  ping-pong kinetics, in which one reactant
  modifies the enzyme, and then a second reactant
  interacts with the modification.

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Bi-substrate Enzyme Kinetics
Sequential 1. ordered 2. random
Ping-pong
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Equations for Bi-substrate Kinetics
B
1/v
VmaxAB
v
KaB KbA AB
1/A
B
VmaxAB
1/v
v
AB KaB KbA KaKb
1/A
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Sequential Kinetics

• Sequential kinetics can be distinguished from
  ping-pong kinetics by initial rate studies.
• In practice, measure initial rates as a function
  of the concentration of one substrate while
  holding the concentration of the second constant.
  Next, vary the concentration of the second
  substrate and repeat.
• Lineweaver-Burk (double-reciprocal) analysis
  should yield a family of lines that intersect at
  the left of the y-axis of the graph.
• Within the realm of sequential reactions lies
  ordered sequential and random sequential at the
  extreme ends. The equations for the two are
  identical therefore, simple initial rate studies
  cannot differentiate between the two.
• In ordered sequential reactions, one substrate is
  obligated to bind to the enzyme before a second
  substrate. In random sequential mechanisms there
  is no preference. In practice, there is usually
  some degree of order in binding.

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Ordered- vs. Random- Sequential
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Adenylate Kinase Kinetic Pathway
Adenylate kinase displays a random ordered
kinetic mechanism. In this case, the two
substrates are bound randomly, and are in
equilibrium with the ternary complex
(EMgATPAMP). As in our derivation, this
necessitates that the off rate for each of the
substrates is less than the forward rate constant
for the chemical step. This allows us to replace
Km with Ks. However, it would not be incorrect
to use Km values. Below is typical shorthand
notation for kinetic schemes.
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Nucleoside Diphosphate Kinase

• Nucleoside diphosphate kinase (NDP Kinase)
  catalyzes the transfer of the terminal phosphoryl
  group of ATP to a nucleoside diphosphate.
• NDP Kinase displays a steady state kinetic
  pattern that is distinctly different from that of
  adenylate kinase. If one substrate is varied
  while the other is fixed at several different
  concentrations, a family of parallel lines is
  obtained by Lineweaver-Burk analysis. This is
  reminiscent of a Ping-Pong reaction.

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Economy in the Evolution of Binding Sites

• Since adenylate kinase and nucleoside diphosphate
  kinase catalyze very similar reactions, why dont
  they proceed by similar mechanisms?
• NDP kinase catalyzes a symmetrical reaction,
  whereas adenylate kinase does not. For NDP
  kinase, the product of the ping (MgADP) is
  similar in structure to the substrate for the
  pong (MgGDP). The only difference involves the
  purine rings of each nucleotide.
• By using a ping-pong reaction, the enzyme can use
  just one binding site for the phsphoryl transfer.
  

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UDP-Glucose Pyrophosphorylase
This is a special type of sequential mechanism in
which MgUTP must bind firs, before
glucose-1-phosphate. There is no degree of
randomness. Ordered binding also implies ordered
product release.
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UDP-Glucose Pyrophosphorylase

• Steady State kinetic equation is similar to
  adenylate kinase. Therefore Lineweaver-Burk
  plots cannot distinguish the two forms of
  sequential reactions. Must do product inhibition
  studies.
• Stereochemistry indicates inversion however,
  incubation of the enzyme with radiolabeled UTP,
  followed by gel-filtration shows a radiolabeled
  intermediate. Be careful! This is because UTP
  or UDP-glucose binds very tightly to the enzyme.
  In fact, the enzyme is isolated with UTP and
  UDP-glucose tightly bound, and will catalyze an
  exchange reaction, which is characteristic of
  Ping-pong reactions.

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Ping-Pong Reaction
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Galactose-1-P Uridylytransferase