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.

2
Phosphoric Acid

• At left are the various ionization states of
  phosphoric acid and the relevant pKa values.
• At pH 7, significant concentrations of inorganic
  phosphate dianion exist. The pKa values are not
  dramatically perturbed in phosphate monoesters
  and phosphate diesters. They would also be
  anionic at physiological pH.
• Phosphate triesters are not common in biological
  systems.
• Frank Westheimer wrote an incredibly insightful
  treatise in 1987, explaining the value of
  phosphate in biochemistry. Its entitled Why
  Nature Chose Phosphates.
• In this treatise, he notes that the fact
  phosphate can link two nucleotides together and
  still ionize (bear a negative charge), thus
  protecting the phosphodiester from hydrolysis.
• In DNA and other biological molecules, the charge
  also ensures that the molecules are retained
  within the lipid membrane.
• The cleavage of phosphoric anhydride bonds
  provide an strong chemical force to drive
  biosynthetic reactions, as will be seen in acyl
  group activation reactions.

3
Examples of Phosphoryl Groups in Biochemistry
4
Small Phosphoryl-Containing Molecules
5
Phosphoryl Amino Acids
6
Classes of Phosphoryl Transfer
In kinases, X is almost always ADP. However, GDP
is known to substitute i some cases.
7
Typical Issues Addressed In Phosphoryl Transfer

• Number and kind of reaction intermediates.
• Does the reaction proceed via covalent catalysis
  (phosphoenzyme intermediate)?
• Does the reaction proceed via direct transfer?
• What is the order of substrate binding?
• Energetics of the reaction.
• What is the rate-limiting transition state?
• How do particular intermediates partition?
• The nature of the elementary step.
• Does phosphoryl transfer proceed via an
  associative transition state?
• Does phorphoryl transfer proceed via a
  dissociative transition state?

8
Dissociative Transition States

• The hydrolysis of phosphomonoester monoanions
  occurs through a dissociative transition state.
• The dissociative transition state is
  characterized by the importance of bond cleavage
  in the transition state.
• There is little bonding to either the attacking
  water molecule or the departing leaving group
  (methanol in the picture on the left) in the
  transition state.
• The minimum requirements for a dissociative
  mechanism is that the phosphoryl group must carry
  two negative charges, and the departing group
  must be a good leaving group.
• The charge density on the phosphorous aids in
  expelling the leaving group, to produce what is
  termed metaphosphate, which can be captured by
  H2O, the acceptor.

actually a double bond. Drawn as such to denote
stereochemistry
9
Associative Transition State

• The hydrolysis of phosphodiester monoanions
  cannot proceed by a dissociative mechanism,
  because the phosphoryl group cannot have the
  double negative charge required for expelling the
  leaving group.
• In solution, phosphodiester anions do react
  (albeit sluggishly) at high temperatures with
  hydroxide ions through an associative transition
  state.
• The associative transition state ic characterized
  by a high degree of bonding between the
  phosphorous and both the entering and leaving
  groups.
• The rate phosphodiester hydrolysis at neutral pH
  is extremely slow, because of the negative charge
  thats born. This is crucial since
  phosphodiester bonds link the nucleotides in DNA

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Pseudorotation

• Notice that in the transition state for an
  associative mechanism, phosphorous is
  pentavalent.
• Unlike carbon, phosphorous is an atom from the
  third row of the periodic table, and can access d
  atomic orbitals to hybridize with its 3s and 3p
  atomic orbitals
• The resulting sp3d hybridization give rise to
  triganol bipyramid geometry, in which three of
  the substituents are equatorial, and lie in a
  plane with an angle of 120C separating each
  bond. The other two substituents are apical, and
  lie above and below the plane.
• Reactions proceeding via associative transition
  states take place with inversion of
  configuration. This necessitates that the
  nucleophile and leaving groups occupy the apical
  positions.
• Pseudorotation refers to the reorganization of
  ligands such that the nucleophile and leaving
  groups occupy the apical positions. This will
  sometimes lead to retention of configuration when
  inversion of configuration is expected.

Crystallographic molecular model of the adduct
between triisopropyl phosphite and
9,10-phenantrhoquinone
11
Cyclic Phosphodiesters

• The five-membered-ring cyclic phosphodiesters are
  hydrolyzed 10 million times as fast as the
  noncyclic or six-membered-ring cyclic analogs.
• Cyclic phosphodiesters react by the extreme
  associative mechanism, in which a discrete
  pentavalent intermediate is formed with single
  bonds linking phosphorous with both the entering
  and leaving groups.
• The driving force for the reaction is that
  formation of the pentavalent species (transition
  state) relieves the bond-angle strain that exists
  in five membered-ring cyclic phosphodiesters.

12
Glucose 6-Phosphatase

• Lets turn to the hydrolysis of glucose
  6-phosphate by the enzyme glucose 6-phosphatase.
• Remember the anomeric carbon when dealing with
  sugars. Unlike many enzymes, this enzyme will
  recognize both anomers of glucose 6-phosphate.
• Questions to address
• Does the reaction proceed with direct attack of
  water at C-6?
• Does the reaction proceed with attack of water at
  the phosphorous?
• Does the reaction take place via covalent
  catalysis?

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Mechanistic Analysis of Glucose 6-Phosphatase

• When the reaction is run in the presence of
  H218O, 18O is found in inorganic phosphate, and
  not in glucose.
• Upon incubation of glucose 6-phosphatase with
  glucose 6-phosphate and 14Cglucose,
  14Cglucose 6-phosphate can be isolated.
• No incorporation of 32P into glucose 6-phosphate
  occurs when the enzyme is incubated with glucose
  6-phosphate and 32P.

14
Isotopic Exchange in Glucose 6-Phosphatase

• Immediate conclusions from our mechanistic
  analysis of the glucose 6-phosphatase reaction
  are the following
• Attack of water clearly does not occur at carbon
  6 to release inorganic phosphate.
• Attack of water takes place on the phosphorous of
  the phosphoryl group.
• Isotope exchange doesnt appear to be mediated by
  direct transfer of inorganic phosphate to
  glucose, since no 32P-labeled glucose 6-phosphate
  when incubated with 32P inorganic phosphate.
• It is unlikely that an enzyme would contain two
  binding sites for glucose if transfer to another
  hexose is not the normal reaction. In fact,
  glucose inhibits competitively versus glucose
  6-phosphate, suggesting that it is occupying the
  same site.
• Isotopic exchange usually suggests a covalent
  enzyme intermediate.

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Mechanism of Isotopic Exchange
16
Isolation of Phosphoryl Enzyme Intermediate
17
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.

18
Adenylate Kinase

• Adenylate kinase was formerly known as myokinase
  because it is found in high concentration 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.

19
Sequential Kinetics

• Sequential kinetics can be distinguished from
  ping-pong kinetics by initial rate studies.
• In practice, measure initial rates as a function
  of one substrate while holding the other
  constant. Then, 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.
• 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
21
Stereochemistry of Sequential Reactions

• The observation of sequential kinetics typically
  indicates a direct transfer of the phosphorous
  group to the relevant nucleophile.
• Direct transfer in an associative mechanism
  suggests that the stereochemistry about the
  phosphorous being attacked should be inverted in
  the product.
• Studies from a number of groups (Benkovic, Frey,
  Eckstein, Knowles etc) led to syntheses of
  chirally-labeled phosphates, which could be used
  to study stereochemistry.
• Problem is that use of sulfur doesnt allow
  stereochemical studies at the terminal
  phosphorous.

22
Use of Oxygen Isotopes
In adenylate kinase, transfer of the terminal
phosphoryl group to AMP proceeds with overall
inversion of configuration at phosphorous. This
is consistent with a single transfer of the
phosphoryl group, as well as a triple
displacement. Almost never see more
displacements than two. Crystal structure of
adenylate kinase bound with Ap5A supports the
single displacement.
23
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.
• In a Ping-Pong reaction, a group is transferred
  from a donor molecule to the enzyme in a first
  half-reaction, which proceeds independently of
  the presence of the acceptor molecule. The group
  is then transferred from the enzyme to the
  acceptor in a second half-reaction.
• The interpretation of Ping-Pong kinetic patterns
  is not always straightforward. The problem is
  that it is often dificult to decide whether
  Lineweaver-Burk plots are really parallel. Also
  various factors can perturb kinetic behavior.

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Isolation of Phosphoryl Enzyme Intermediate

• In contrast to what weve seen with glucose-6
  phosphatase, the isolation of a phosphoryl enzyme
  intermediate in the case of ping-pong reactions
  should be relatively straightforward. The trick
  is to incubate the enzyme with the first
  substrate only. Cant do this with glucose-6
  phosphatase, since the second substrate is water.
  
• After incubating with the first substrate,
  unbound molecules can be separated form the
  protein by gel-filtration chromatography.
• Subsequent to isolating the protein fraction, and
  total hydrolysis under alkaline conditions, a
  phosphohistidine amino acid was isolated.
• Stereochemical analysis of he reaction using
  18Othiophosphoryl analog of ATP results in
  retention of configuration about the phosphorous.
  This is consistent with a double displacement
  reaction. One displacement takes place via the
  enzyme histidine, and the second displacement
  takes place with water.

25
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.
  

26
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
29
Galactose-1-P Uridylytransferase
30
Determination of Kinetic Pathways in
Multisubstrate Enzymatic Reactions

• Clelands rules enable one to deduce the types of
  enzyme-inhibitor binding interactions that lead
  to inhibition simply by inspecting the plots of
  1/v versus 1/(varied substrate) obtained in the
  presence and absence of the inhibitor.
• The inhibitor may be an unreactive molecule
  related to the substrate by structure, or it may
  be a reaction product that binds to the enzyme in
  place of a substrate and reverses the flow of
  chemical events in catalysis.
• When the inhibitor is a reaction product, its
  interaction with the enzyme gives an inhibition
  pattern that is characteristic of the kinetic
  pathway for enzyme-substrate and enzyme-product
  binding and these inhibition patterns for
  several inhibitors can be useful for deducing the
  kinetic binding pathway.

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Clelands Rules

• Rule 1 An inhibitor that binds to the same
  enzyme form as does the varied substrate, or to a
  form that is connnected only by reversible steps
  to that form, increases the slope of the
  Lineweaver-Burk plot.
• Rule 2 An inhibitor that binds to a different
  enzyme form from that to which the varied
  substrate binds increases the intercept of the
  double-reciprocal plot.
• Thus, an inhibitor that binds only according to
  rule 1 will affect only the slopes in the
  double-reciprocal plot, and this corresponds to
  competitive inhibition. An inhibitor that binds
  only according to rule affects only the
  intercepts (lines will be parallel), and this
  corresponds to uncompetitive inhibition. An
  inhibitor that binds in accord with both rule 1
  and 2 exhibits both slope and intercept effects.
  This is noncompetitive inhibition, and the lines
  will converge at the left of the ordinate.