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Nucleophilic Substitution at Phosphorus

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									Nucleophilic Substitution at
      Phosphorus




    David J. Weber, PhD
   dweber@umaryland.edu
 Classes of nucleophilic substitution
                            NEVER




Knowles, 1980
              Nucleophilic attack of Y:
 Attack at:
 Alpha




 Beta




 Gamma



Leaving group is either PPi, AMP, or ADP
Associative vs
Dissociative

Experimental
Criteria:

1. Stereochem
2. Basicity
3. Entropy
4. Product ratio
5. Isotope effect
6. Effect of S
7. Gen Base
8. Others
ATP Rxns
Location of
metal
Rxn coordinate
Stereochem at
Phosphorus
General Base
Isotope effects
Enzymes for today
Staphylococcal Nuclease




RXN catalyzed – DNA hydrolysis
Compare bound pdTp conformation on
       SN by NMR & X-ray




pdTp is transition state analogue and highly
competitive inhibitor (support Assoc Mech)
NMR review

        Paramagnetic
        metals such as
        Mn2+ or Co2+ etc
        cause 1/T1
        relaxation rates
        to go faster as a
        f(distance;1/r6)
        from nuclei
Relaxation rate
depends on
distance and:

1. Occupancy of
   metal in binary
   vs. ternary
   state

2. Outer sphere
   effects

3. Paramagnetic
   nature of metal

4. Correlation
   time
   Paramagnetic relaxation data from a
    SN bound substrate, dTdA  two
        possible conformations




Distances are consistent with two conformations
 that are analogous to having an umbrella in the
       normal and inverted conformations
NOE and 1/T1 data of dTdA


               With long-range
               and short-range
               NOE data there is
               only 1 possible
               conformation
               (anti, anti).
Why is Staph Nuclease so fast?
                   16 orders of
                   magnitude of rate
                   acceleration

                   It approximates the
                   reactants

                   3D walk versus no
                   walk
Why is Staph Nuclease so fast?




                        Answer #2:
                        Enzymes
                        stabilize
                        the
                        transition
                        State
Why is Staph Nuclease so fast?


Answer #2:
Enzymes
stabilize
the
transition
State
                          Knowles’ General
                         Phosphoryl Transfer
                            Mechanism
     Positive charge or H-bond




          General Acid                 General Base




Transition state for in-line displacement requires charge
stabilization & general acid/base from the enzyme
Why is Staph Nuclease so fast?
                         Answer #3:
                         General
                         acid/base




                        Weber et al.,
                        (1992) Proteins
                        13, 275
 Is E43 really a general base?

                                 Answer:
                                 Yes!




Even though the E43S is 1000-fold less
active, it is more sensitive to pH (i.e. base)
Modify residues in the active site
                          Chemical
                          Modification
                          or Mutation

                          Causes an
                          increase in
                          free energy
                          typically
   Modify two residues in the
           active site


Different              Same
steps                  step
affected               affected




Modifications can affect the same step or
different steps  need to determine
mechanism of mutant enzyme
    Results of double mutant
 studies acting on the same step
                                     R87G  ~5 orders of
                                            magnitude

                                     E43S  ~4 orders of
                                            magnitude

                                     DM    ~6 orders of
                                           magnitude
                                           (not 9 orders of mag)




Partially additive effects for mutations of
the general acid/base indicate that these
residues cooperatively bind the t.s.          Weber et al., (1992)
                                              Biochem. 30, 6103
General acid and general base
  cooperate to bind the t.s.
                        Answer #3:
                        General
                        acid/base




                       Weber et al.,
                       (1992) Proteins
                       13, 275
                          Knowles’ General
                         Phosphoryl Transfer
                            Mechanism
     Positive charge or H-bond




          General Acid                 General Base




Transition state for in-line displacement requires charge
stabilization & general acid/base from the enzyme
simultaneously and together they stabilize the t.s.
     Exonuclease of DNA Pol I
Reaction catalyzed – DNA hydrolysis
DNA synthesis fidelity – 1 error in 109 to 1010
   Some enzymes aren’t as evolved as
           Staph Nuclease




Don’t want a “hot” nuclease in
DNA pol I  kcat makes sense


   kcat is about 1000-fold lower than staph
   nuclease (95/sec) as for E43S mutant 
   no general base as in staph nuclease
 Catalytic RNA doesn’t have a
     general base either




Self-splicing mechanism for which Tom
Cech won the Nobel Prize
One pathway for self-slicing
                      Two steps 
               exon   GOH attack
        exon          followed by
                      intramolecular
                      attack

                      Exon strands
                      are in lower
                      case letters
RNA hydrolysis by catalytic RNA
   Let’s take a closer look at
           ribozymes




Mg2+ is required (10-20 mM); Mn2+ also
works
Evidence for Mg2+ stabilizing T.S. charge




    Substrate (S) 




Mg2+ -enzyme prefers O and Cd2+-E prefers S at
position X of S; therefore, metal located as shown
Here is the data   Mg2+ prefers O
                   and Cd2+
                   prefers S at
                   position X;
                   therefore,
                   metal located
                   as shown
            Hexokinase (yeast)
  Rxn 

ATP + D-glucose  ADP + D-glucose 6 phosphate + H+

 - irreversible under intracellular conditions
 - priming reaction for the entry into glycolysis
Hexokinase (yeast)
                     k1/k-1 = 661
                     and favors
                     G6P to be
                     maximal for
                     further
                     metabolism

                     KM=0.1mM
                     for ATP
   Side
Reaction
for Yeast
  HK is
 avoided
 without
 glucose
Conformational change explains
     substrate synergism
          open




         closed




 When glucose binds a conformational
 change increases ATP binding and
 enhances catalytic conversion to G6P
       Hexokinase (close-up)



                               Co2+ (not Mg2+)
                       β

                          




Evidence from X-ray that attack is at gamma;
probably long b/c since it is an inhibitor (not S)
Product binding is in substrate
     site of hexokinase




           Transition state for
           associative mechanism
           has overlapping substrate
           and product in active site
          Pyruvate Kinase
 Rxn  Last step in glycolysis:

 phosphoenolpyruvate + ADP 

 enolpyruvate + ATP

Note:
Enol pyryvate  ketopyruvate (at pH 7)
Pyruvate Kinase (230 kDa tetramer;
       57 kDa per subunit)




Enzyme favors ATP synthesis (Keq = 3000)
Pyruvate is major metabolite, but on PK 
enolate forms
                               PK
                             reaction
                                in
                            glycolysis



Tritium exchange observed only when Pi
present  conformational change required
Structural
 data for
   PK
Results of all the PK structures




Model consistent with all the structures
calculated
Products and Substrates Overlap




 Product inhibition observed and is also
 consistent with an associative reaction
cAMP-dependent protein kinase
            Rxn catalyzed 
           phosphorylase kinase
              (inactive)
                    + ATP
          
          Phosphorylase kinase-P
              (active)
          + ADP
Stimulation
of glycogen
breakdown
cAMP-dependent protein kinase




1/T1
methods                                 NOEs




  ATP-bound structure completed first
      cAMP-dependent kinase



              ATP




  Ser that is
  phosphorylated




Peptide substrate is in extended conformation
Close-up view of cAMP-kinase




Structures all consistent with attack at  P
when cAMP binds
        PRPP synthetase




                PRPP

PRPP – 5-phosphoribosyl-1-pyrophosphate
  PRPP
synthetase
  (cont)



1st enzyme
shown to have
attack at β-
phosphate
Geometry for PRPP synthetase

    
                                           CD

        β
            



Attack at β-P with inversion of configuration
is consistent with all the data available
DNA polymerase – attack at 




Structure of ATP in the binary enzyme
complex and inversion of configuration
confirmed with alpha-S-TTP experiments
X-ray and NMR data   Bound
                     substrates
                     and DNA are
                     both in B-
                     DNA form
                     and not in A
                     or Z form
Reaction can only proceed when
 template and primer are bound




Correct base pair is necessary
Close-ups of DNA polymerase




Correct base pair is necessary

								
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