Catalysis

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					Enzyme Catalysis

    3/17/2003
     General Properties of Enzymes

•Increased reaction rates sometimes 106 to 1012 increase
      Enzymes do not change DG just the reaction rates.
•Milder reaction conditions
•Great reaction specificity
•Can be regulated
              Substrate specificity
The non-covalent bonds and forces are maximized to
bind substrates with considerable specificity
•Van der Waals forces
•electrostatic bonds (ionic interactions)
•Hydrogen bonding
•Hydrophobic interaction
                            enz
                   A+B            P+ Q
              Substrates           Products
          Enzymes are Stereospecific
                            O
                                       
CH 3CH 2 OH  NAD  CH 3CH  NADH  H

        Yeast
Alcohol dehydrogenase
                          O
                      H
                          C         NAD+
                              NH2

  CH3CD2OH +
                      +
                      N   Ox.
                                    D           O
                                            H
                     O                          C
                                                    NH2
                 CH3C-D       +
                                        N       Red.

                                    NADD
Pro-R hydrogen gets pulled off

       Yeast Alcohol dehydrogenase
                                             OH
              O
2. NADD + CH3-C-H                   H        C         D


                                             CH3

         O                                       OH
3. CH3-C-D + NADH
                                        D        C         H


                                                 CH3
If the other enantiomer is used, the D is not transferred

YADH is stereospecific for Pro-R abstraction
Both the Re and Si faced transfers yield identical
products. However, most reactions that have an Keq
for reduction >10-12 use the pro-R hydrogen while
those reactions with a Keq <10-10 use the pro-S
hydrogen. The reasons for this are still unclear
    Specific residues help maintain
            stereospecificity

Liver alcohol dehydrogenase makes a mistake
 1 in 7 billion turnovers. Mutating Leu 182 to
Ala increases the mistake rate to 1 in 850,000.
This is a 8000 fold increase in the mistake rate,
    This suggests that the stereospecificity is
        helped by amino acid side chains.
           Geometric specificity
    Selective about identities of chemical groups
                        but
    Enzymes are generally not molecule specific
There is a small range of related compounds that will
            undergo binding or catalysis.
Similar shaped molecules can be highly toxic

     O
H
     C
         NH2

                                    N
 +
 N                                   CH3
                    N

                        Tobacco Nicotine

         Because of this closeness in name
Nicotinic acid was renamed to niacin by the bread
                  manufactures
                     Coenzymes
Coenzymes: smaller molecules that aid in enzyme chemistry.
Enzymes can:
     a. Carry out acid-base reactions
     b. Transient covalent bonds
     c. Charge-charge interactions
Enzymes can not do:
     d. Oxidation -Reduction reactions
     e. Carbon group transfers

Prosthetic group - permanently associated with an enzyme
or transiently associated.
Holoenzyme: catalytically active enzyme with cofactor.
Apoenzyme: Enzyme without its cofactor
                   Commom Coenzymes
Coenzyme                   Reaction mediated
Biotin                      Carboxylation
Cobalamin (B12)             Alkylation transfers
Coenzyme A                  Acyl transfers
Flavin                      Oxidation-Reduction
Lipoic acid                       Acyl transfers
Nicotinamide                Oxidation-Reduction
Pyridoxal Phosphate         Amino group transfers
Tetrahydrofolate            One-carbon group transfers
Thiamine pyrophosphate      Aldehyde transfer
             Vitamins are Coenzyme precursors

Vitamin              Coenzyme               Deficiency Disease
Biotin                Biocytin                    not observed
Cobalamin (B12)      Cobalamin             Pernicious anemia
Folic acid           tetrahydrofolate      Neural tube defects
                                           Megaloblastic anemia
Nicotinamide         Nicotinamide          Pellagra
Pantothenate         Coenzyme A                   Not observed
Pyridoxine (B6)      Pyridoxal phosphate          Not observed
Riboflavin (B2)      Flavin                       Not observed
Thiamine (B1)        Thiamine pyrophosphate       Beriberi
These are water soluble vitamins. The Fat soluble
vitamins are vitamins A and D.
Humans can not synthesize these and relay on their
presence in our diets. Those who have an unbalanced diet
may not be receiving a sufficient supply.
Niacin (niacinamide) deficiency leads to pellagra
characterized by diarrhea, dermatitis and dementia.
Pellagra was endemic is Southern United States in the
early 20th century. Niacin can be synthesized from the
essential amino acid, tryptophan. A corn diet prevalent at
the time restricted the absorption of tryptophan causing a
deficiency. Treatment of corn with base could release the
tryptophan (Mexican Indians treated corn with Ca(OH)2
before making tortillas!)
        Regulation of Enzymatic Activity
There are two general ways to control enzymatic activity.
      1. Control the amount or availability of the enzyme.
      2. Control or regulate the enzymes catalytic activity.
Each topic can be subdivided into many different
categories.
Enzyme amounts in a cell depend upon the rate in which it
is synthesized and the rate it is degraded. Synthesis rates
can be transcriptionally or translationally controlled.
Degradation rates of proteins are also controlled.
However, We will be focusing on the regulation of
enzymatic activity.
The catalytic activity of an enzyme can be altered
either positively (increasing activity) or negatively
(decreasing activity) through conformational
alterations or structural (covalent) modifications.
Examples already encountered is oxygen, carbon
dioxide, or BPG binding to hemoglobin.
Also, substrate binding to the enzyme may also be
modified by small molecule effectors changing its
catalytic site.
Protein phosphorylation of Ser residues can activate
or deactivate enzymes. These are generally
hormonally controlled to ensure a concerted effect on
all tissues and cells.
            Aspartate Transcarbamoylase:
             the first step in pyrimidine biosynthesis.
                                      -                            O-
                                  O                      O
                       O
                                                               C
                           C
    NH2                                    ATCase                  CH2
                                  CH2                    NH2
                                                                         + H2PO4-
    C              +                                               C
          OPO3--                  C                      C
O                                                                  H     COO-
                                  H             -   O          N
                                          COO
                           H3N+                                H


Carbamoyl                      Aspartate                N-Carbamoyl aspartate
phosphate

This enzyme is controlled by Allosteric regulation and
                Feedback inhibition
Notice the S shaped curve (pink) cooperative binding of aspartate
Positively homotropic cooperative binding
Hetertropically inhibited by CTP
Hetertropically activated by ATP
Feedback inhibition
Where the product of
a metabolic pathway
inhibits is own
synthesis at the
beginning or first
committed step in the
pathway
CTP is the product of this pathway and it is also a
precursor for the synthesis of DNA and RNA (nucleic
acids). The rapid synthesis of DNA and/or RNA
depletes the CTP pool in the cell, causing CTP to be
released from ATCase and increasing its activity. When
the activity of ATCase is greater than the need for CTP,
CTP concentrations rise rapidly and rebinds to the
enzyme to inhibit the activity.
ATP activates ATCase. Purines and Pyrimidines are
needed in equal amounts. When ATP concentrations
are greater than CTP, ATP binds to ATCase activating
the enzyme until the levels of ATP and CTP are about
the same.
Enzymatic catalysis and mechanisms
•A. Acid - Base catalysis
•B. Covalent catalysis
•C. Metal ion aided catalysis
•D. Electrostatic interactions
•E. Orientation and Proximity effects
•F. Transition state binding
General Acid Base
Rate increase by partial proton abstraction by a
Bronsted base or
Rate increase by partial proton donation by a
Bronsted Acid
 Mutarotation of glucose by acid and base catalysts




         d   D  glucose
                             k   D  glucose
      v                        obs             
                  dt
The reaction can be followed by observation of the optical activity
                             change
Kobs = apparent first order kinetics but increases with
increased concentrations of acid and base.

                                  The acid HA donates a
                                  proton to ring oxygen,
                                  while the base abstracts a
                                  proton from the OH on
                                  carbon 1. To form the
                                  linear form. The cycle
                                  reverses itself after
                                  attacking the carbonyl
                                  from the other side.
This compound does not undergo mutarotation in
aprotic solvents. Aprotic solvents have no acid or
base groups i.e. Dimethyl sulfoxide or dimethyl
formamide. Yet the reaction is catalyzed by
phenol, a weak acid and pyridine, a weak base

 v = k[phenol][pyridine][TM--D-glucose]
The reaction can be catalyzed by the addition of
             -Pyridone as follows




     v=k'[-pyridone][TM-a-D-glucose]
 k' = 7000M x k or 1M -pyridone equals
    [phenol]=70M and [pyridine]=100M
    Many biochemical reactions require acid
               base catalysis
•Hydrolysis of peptides
•Reactions with Phosphate groups
•Tautomerizations
•Additions to carboxyl groups
Asp, Glu, His, Cys, Tyr, and Lys have pK’s near
physiological pH and can assist in general acid-base
catalysis.
Enzymes arrange several catalytic groups about the
substrate to make a concerted catalysis a common
mechanism.
RNase uses a acid base mechanism
Two histidine residues catalyze the
reaction. Residue His 12 is deprotonated
and acts as a general base by abstracting
a proton from the 2' OH.
His 119 is protonated and acts as a
general acid catalysis by donating a
proton to the phosphate group.
The second step of the catalysis His 12
reprotonates the 2'OH and His 119 reacts
with water to abstract a proton and the
resulting OH- is added to the phosphate.
This mechanism results in the hydrolysis
of the RNA phosphate linkage.
           Covalent catalysis

  Covalent catalysis involves the formation of a
transient covalent bond between the catalyst and
                  the substrate
 Catalysis has both an nucleophilic and an electrophilic stage
1 Nucleophilic reaction forms the covalent bond
2 Withdrawal of electrons by the now electrophilic catalyst
3 Elimination of the catalyst (almost the reverse of step 1)
  Depending on the rate limiting step a covalent
   catalytic reaction can be either elecrophilic or
nucleophilic. Decarboxylation by primary amines
 are electrophilic because the nucleophilic step of
         Schiff base formation is very fast.
    Nucleophilicity is related to the basicity but
instead of abstracting a proton it attacks and forms
                  a covalent bond.
Lysines are common in formation of schiff bases while
 thiols and imidazoles acids and hydroxyls also have
     properties that make good covalent catalysts
 Thiamine pyrophosphate and pyridoxal phosphate
             also show covalent catalysis
                Metal ion catalysts
One-third of all known enzymes needs metal ions to work!!
1. Metalloenzymes: contain tightly bound metal ions: I.e.
Fe++, Fe+++, Cu++, Zn++, Mn++, or Co++.
2. Metal-activated enzymes- loosely bind ions Na+, K+,
Mg++, or Ca++.
          They participate in one of three ways:
      a. They bind substrates to orient then for catalysis
      b. Through redox reactions gain or loss of electrons.
      c. electrostatic stabilization or negative charge
         shielding
Charge stabilization by metal ions


             Metal ions are effective
             catalysts because unlike
             protons the can be
             present at higher
             concentrations at
             neutral pH and have
             charges greater than 1.
    Metal ions can ionize water at higher
               concentrations

  The charge on a metal ion makes a bound water more
  acidic than free H2O and is a source of HO- ions even
                      below pH 7.0

    NH3 5 Co3 H2O  NH3 5 Co3 OH-  H

The resultant metal bound OH- is a potent nucleophile
    Carbonic Anhydrase

                  -    
CO 2  H 2O  HCO3  H
Charge shielding
     Proximity and orientation effects




 
d p  NO2O               
               k1imidizole p  NO2Ac  k1p  NO2Ac
                                            
       dt
   k'1 = 0.0018s-1 when [imidazole] = 1M




   When the phenyl acetate form is used
           k2 = 0.043 or 24k'1

Proximity effects lead to relatively small rate
               enhancement!
•Reactants are about the same size as water molecules
      (approximation)
•Each species has 12 nearest neighbors (packed spheres)
•Reactions only occur between molecules in contact
•Reactant conc. Is low so only one can be in contact at a
      time
                        dA B
                                k1AB  k 2 A, Bpair
         k1
A  B  A        B v
                           dt
                              12AB
                A, Bpairs 
                               55.5M

       v  k1
              55.5
                   A, Bpairs  4.6k1A, Bpairs
               12
Only a 4.6 rate enhancement but molecular motions
if slowed down leads to a decrease in entropy and
rate enhancements.


Molecules are not as reactive in all directions and
many require proper orientation to react. Increases
in rates of 100 fold can be achieved by holding the
molecules in their proper orientation for reaction.
 Preferential transition state binding
Binding to the transition state with greater affinity to
           either the product or reactants.
                  RACK MECHANISM

              Strain promotes faster rates
  The strained reaction more closely resembles the
 transition state and interactions that preferentially
   bind to the transition state will have faster rates
        kN                   kN for uncatalyzed reaction
      
   S   P
                                          and
         kE
     
 ES  EP                     kE for catalyzed reaction
               K N‡    ‡
             
     E  S   S  E  P  E
        KR                 KT                 
                  ‡
               KE               ‡
          
      ES   ES                               EP
                                                     ‡
       ES            ES ‡        SE N
                                            ‡
 KR             KT 
                                             K
      ES          ES‡        SE

     ES       K T SES  K E
 ‡      ‡                               ‡                ‡
KE 
      ES          ‡      
                K R S ES K N ‡
                 k BT  ‡  k B T  ‡
v N  k N S         S       K N S
                 h             h 


      k BT  ‡  k BT  ‡
vE         ES      K E ES
      h              h 
                     ‡
           k E K E KT
                 ‡
           k N K N KR
Preferential transition state binding
The more tightly an enzyme binds its reaction’s
transition state (KT) relative to the substrate
(KR) , the greater the rate of the catalyzed
reaction (kE) relative to the uncatalyzed
reaction (kN)


Catalysis results from the preferred binding
and therefore the stabilization of the transition
state (S ‡) relative to that of the substrate (S).
         DG N  DGE       106 rate enhancement
kE       
                      RT 
                          
    exp                      requires a 106 higher
kN                            affinity which is 34.2
                              kJ/mol
The enzyme binding
of a transition state
(ES‡ ) by two
hydrogen bonds that
cannot form in the
Michaelis Complex
(ES) should result in
a rate enhancement
of 106 based on this
effect alone
Transition state analogues are competitive
                 inhibitors

				
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