100805Catalysis by qihao0824

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									Enzyme chemistry of nucleophilic oxy anion and enzyme-coenzyme chemistry of electron and group
transfer

I. Purpose and principle of enzyme catalysis: 1. Why biocatalysis is needed and how it is done. 2. What
enzymes can and cannot do. 3. Shape fitting a prerequisite to enzyme specificity and catalysis. 4. A
practical and revealing description of enzyme kinetics. 5. Kinetics of an allosteric enzyme that shows
cooperativity. 6. Chemical background of catalysis by general-acid/base and metal ion.

II. Enzyme chemistry of nucleophilic oxy anion: 1. Serine protease oxy anion as nucleophile. Triad
catalytic strategy is an especially effective approach to peptide hydrolysis, Structure of chymotrypsin,
Selective peptide cleavage, Chymotrypsin and related hydrolytic enzymes (trypsin, elastase) and their
substrate specificities, Papain S¯ in lieu of O¯ as nucleophile. 2. Aspartyl protease of HIV and protease
inhibitor as AIDS drug. Mechanism of specific inhibition of HIV protease. 3. Carboxypeptidase A
cleaves peptide bond next to C-terminal-COO¯ using Zn++. 4. More Zn++ ion catalysis and synthetic
model of carbonic anhydrase. 5. Lysozyme hydrolyzes bacterial cell wall composed of β-glycosidic
linkages. 6. Isomerization of dihydroxyacetone phosphate to glyceraldehyde 3-phosphate in glucose
catabolism. 7. Bacterial transpeptidase. Penicillin inactivates an enzyme-serine in bacterial cell-wall
synthesis.

III. Enzyme-coenzyme chemistry of electron transfer (NAD+ and FAD): 1. Coenzyme and vitamins for
electron transfer and structural link. 2. Structures of NAD+/NADH & niacin vitamin for redox reactions.
3. Glyceraldehyde 3-phosphate dehydrogenase and NAD+. NAD+-binding region in dehydrogenases,
Catalytic mechanism, Free energy profiles for glyceraldehyde oxidation coupled/uncoupled with acyl-
phosphate formation. 4. Flavin redox coenzymes FAD/FADH2 are covalently bound to enzyme and
complimentary to NAD/NADH. Mechanism of dehydrogenation, Why FAD as another electron carrier
and why not use NAD+ throughout, FAD precedes NAD+ in major metabolic pathways.

IV. Enzyme-coenzyme chemistry of group transfer (Imine chemistry of pyridoxal and cryptic carbanion
chemistry of thiamin): 1. Coenzyme and vitamins for group transfer. 2. Pyridoxal in group transfer
reactions. Structures of pyridoxal (free and enzyme bound) and reactivity, Pyridoxal mediate group
transfers, Mechanism via imine-enamine intermediates (transamination, decarboxylation, transfer of
RCHO, transfer of -H makes L-amino acids), Seleative bond cleavage by stereoelectronic control of
PLP Enzymes. 3. Cryptic carbanion chemistry of thiamin. Structure of thiamin and unique ylidene as
carbanion, Significant role of thiamin in mediating between glycolysis and the citric acid cycle, pyruvate
dehydrogenase complex joins with thiamin for crucial link, Mechanism via thiamin ylidene intermediate
in decarboxylation and transacetylation for the dehydrogenase system, Schematic diagram of the dimer
interface environment around the catalytic center, Illustration of complete reaction schemes of the
dehydrogenase complex, Detailed picture of the transketolase-thiamin-substrate, Fundamental structural
questions resolved.
I. Purpose and principle of biocatalysis

1. Why biocatalysis is needed. Fast reactions are required to sustain life. For example, the half-life for
chemical hydrolysis of a peptide bond at neutral pH is 10 - 1000 years. Yet, peptide bonds must be
hydrolyzed within milliseconds in some biochemical processes. See the table of rate enhancement
achieved as enzyme catalysis has evolved.

Rate enhancement by selected enzymes

Enzyme                        Nonenzymatic           Rate enhancement
                                 half-life                (kcat/kun)
OMP decarboxylase            78,000,000 years            1.4 × 1017
Staphylococcal                130,000 years              5.6 × 1014
nuclease
Carboxypeptidase A                7.3 years                1.9 × 1011
Triose phosphate                  1.9 days                 1.0 × 109
isomerase
Carbonic anhydrase               5 seconds                  7.7 × 106
(OMP, orotidine monophosphate)

A. Radzicka and R. Wofenden. Science 267 (1995):90 93.

 How it is done. The enzymes (or with coenzymes) help to form and stabilize the transition state. The
basic strategies are: (1) Bring reacting groups together in the active site, properly oriented, to achieve
specificity. (2) Exert binding energy to stabilize the TS. (3) Form more active intermediate, e.g., amide to
acid product via ester. (4) Use side chains of amino acids for general acid-base catalysis. (5) Metal ion,
when bound to enzyme, participates in coordination and as a Lewis acid. (6) Make use of coenzymes for
H¯ and C¯ involvement.


Six major classes of enzymes

Class              Type of reaction                                        Example
1. Oxidoreductases Oxidation-reduction                                     Lactate dehydrogenase
2. Transferases    Group transfer                                          Nucleoside monophosphate
                                                                           kinase
3. Hydrolases       Hydrolysis reactions (transfer of functional groups    Chymotrypsin
                    to water)
4. Lyases           Addition or removal of groups to form double bonds Fumarase
5. Isomerases       Isomerization (intramolecular group transfer)       Triose phosphate isomerase
6. Ligases          Ligation of two substrates at the expense of ATP    Aminoacyl-tRNA synthetase
                    hydrolysis
          2. What enzymes can and cannot do

          Enzymes alter only the reaction rate and not the equilibrium.

          Consider




          The equilibrium concentration of B is 100 times that of A, whether or not enzyme is present. Enzyme
          accelerates both forward and reverse rates, hence attaining equilibrium faster but do not shift its K.
          K = function of ∆G between reactants and products.

Catalysis What enzymes can do: facilitates TS formation by lowering its activation energy




                                                                              TS energy lowered by enzyme cat.


Catalyst destabilizes the transition state
 entice Hall 2001                         Chapter 22                                                   4
3. Shape fitting a prerequisite to enzyme specificity and catalysis

Much of the catalytic power of enzymes comes from bringing substrates together in
optimal orientations at the active site. Formation of the enzyme-substrate (ES) complexes
is a prelude to making and breaking bonds at TS.

Enzymes contain >100 amino acids, MW >10 kd and a diameter of >25 Å. The vast
majority of amino acids serve as a scaffold to create the 3-D active site, a pocket, from a
few amino acids that may be far apart in the primary structure.

Enzymes are flexible and adopt conformations that are structurally and chemically
complementary to the transition states of the reactions that they catalyze. Thus, other than
lock and key fit, shape fitting of ES complex may be induced.




Substrates are bound in the active site by multiple weak attractions: electrostatic, H-bonds,
van der Waals, and hydrophobic forces to give ES complexes with K that range from 10 -2
to 10-8 M. This binding energy of the ES complex helps to stabilize the TS.
4. A practical and revealing description of enzyme kinetics

A plot of the reaction velocity (V0) as a function of the substrate concentration [S] for an enzyme shows
the maximal velocity (Vmax) is approached asymptotically. Note KM, a constant, is the [S] yielding a
velocity of Vmax/2. KM lies between 10-1 and 10-7 M, pointing to [S] required for significant catalysis to
occur. KM is also a measure of the strength of the ES complex: a high KM indicates weak binding; a low
KM indicates strong binding.




This behavior is summarized in the Michaelis-Menten equation:



Substrate concentration when low, [S] << KM, V0 = (Vmax/KM)[S]; rate is directly
proportional to [S]. When high, [S] >> KM, V0 = Vmax; rate is maximal, independent of [S].
Physiological consequence of KM: Consider the sensitivity of some individuals to ethanol. Such
persons exhibit facial flushing and rapid heart rate after ingesting even small amounts of alcohol. In the
liver, CH3CHO is the cause of intoxication, then [O] to CH3COO¯ by acetaldehyde dehydrogenase.




Most people have 2 forms of the acetaldehyde dehydrogenase, a low KM mitochondrial
form and a high KM cytosolic form. In susceptible persons, the mitochondrial enzyme is
less active due to a single amino acid mutation, leaving CH3CHO to the cytosolic enzyme.
Because this enzyme has a high KM, less CH3CHO is converted into CH3COO¯; excess
CH3CHO escapes into the blood and accounts for the physiological effects.
Meaning of Vmax: The maximal rate reveals the turnover number, which is the number of
substrate molecules converted into product by an enzyme molecule in a unit time.
e.g., A 10-6 M solution of carbonic anhydrase catalyzes the formation of 0.6 M H2CO3 s-1
when fully saturated with CO2. Hence, turnover is 6 × 105 s-1, one of the largest known.
The opposite is 0.5 s-1 for lysozyme, but most enzymes fall in the range from 1 - 104 s-1.
5. Kinetics of an allosteric enzyme that shows cooperativity

Allosteric enzymes display a sigmoidal dependence of reaction velocity on [S].




Here the binding of substrate is cooperative (binding of substrate to one active site
facilitates substrate binding to the other active sites).

Allosteric effect of enzyme

Consider hexokinase which binds glucose in the first step of glycolysis. The result of this
step is to “prime” glucose for subsequent breakdown by attaching a phosphate to it. This
phosphate comes from ATP, which binds to hexokinase after the binding of glucose
causes a change in the enzyme’s shape. Thus, hexokinase can exist in two forms,
depending on what is bound. This is allosterism or “other forms”. In this case, the
allosteric change is one that activates the enzyme.




Another allosteric scheme is (1) the change from binding ATP, and (2) the next change when the ATP is
hydrolyzed. This scheme is the motor driving the muscle protein, myosin, and ion pumps.

The allosteric enzyme activity may be altered by regulatory molecules bound to sites other than the
catalytic sites, e.g., hemoglobin binding O2 is cooperative and is regulated by H+, CO2 etc.
        6. Chemical background of catalysis

          Electron push and pull most effective




cid Catalysis
If the proton transfer occurs during the
                          is known
slow step, it catalyzed hydrolysis of ester as general-acid
     Chemical examples of
catalysis and involves a weak acid, HA
             Intramolecular Catalysis
          General-acid catalysis


              Putting the reacting group and the
               catalyst on the same molecule has an
               effect similar
          General-base catalysis to placing both reacting
               groups on the same molecule                          Intramolecular Cata
          Ortho-carboxyl as intramolecular general-base catalyst: aspirin susceptible to moisture
ice Hall 2001                                Chapter 22                                                             12
                                                                                                                     The or
                                                                                                                     substit
                                                                                                                     intramo
             Acid Catalysis                                                                                          genera
                                                                                                                     that inc
                                                                                                                     nucleo
           © Prentice Hall 2001          Chapter 22                          25
                                                                                                                     water
                                                                                 This is called intramolecu
                                                                                  catalysis or anchimeric a
                                                                             © Prentice Hall 2001             Chapter 22




        Specific cat. (activated species preformed before TS) is more for chemical than bio reactions.



          © Prentice Hall 2001                        Chapter 22                                         13
                            Metal-Ion Catalysis
                                A metal ion can

    Metal ion catalysis                   stabilize a
                                          transition state byA metal ion can
                                                                  

                                          receiving                  make a leaving
                                          electrons from a group a weaker
                                          center where a base
                                          negative charge
                                          is developing
    Metal-Ion Catalysis
     TS stabilized                  Leaving tendency of OCH3 enhanced


  © Prentice Hall 2001             Chapter 22                           18




                          © Prentice Hall 2001             Chapter 22                 19




                                      Better nucleophile
   A metal ion can complex with water
  Water becomes more acidic when metal-bound, yielding an effective HO¯
    and increase its acidity

© Prentice Hall 2001                Chapter 22                               20
II. Enzyme chemistry of nucleophilic oxy anion

1. Serine protease oxy anion nucleophile: Triad catalytic strategy is an especially effective approach
   to peptide hydrolysis.

Catalytic triad found in chymotrypsin: Asp 102 side chain induces His 57 to remove H+
from Ser 195, thus activating it to Ser–CH2O¯ to initiate peptide hydrolysis.




Structure of chymotrypsin (endopeptidase). 3 chains are shown in ribbon form in orange,
blue, and green. The side chains of the catalytic triad residues, including serine 195,
unusually reactive, are shown. Chymotrypsin is inactivated by treatment with
diisopropylphosphofluoridate (DIPF), which reacts only with serine 195 among 28
possible serine residues.
Selective peptide cleavage

Chymo cleaves peptide bonds selectively on the carboxyl terminal side of the large
hydrophobic amino acids such as try, tyr, phe, and met.




 Hydrolysis takes place in two stages: (A) acylation to form the acyl-enzyme intermediate
followed by (B) deacylation to regenerate the free enzyme.
Chymotrypsin and related hydrolytic enzymes (trypsin, elastase) and their substrate specificities

The sequences of trypsin and elastase are approximately 40% identical with that of
chymotrypsin, and their overall structures are nearly the same.




An overlay of the structure of chymotrypsin (red) on that of trypsin (blue) shows the high
degree of similarity. Only a-carbon atom positions are shown. The mean deviation in
position between corresponding a-carbon atoms is 1.7 Å.

Different substrate specificities: Trypsin cleaves peptide bond after residues with long,
positively charged side chains (Arg, Lys). Elastase cleaves peptide bond after amino acids
with small side chains (Ala, Ser).


Papain S¯ in lieu of O¯ as nucleophile




Papain, an enzyme from the papaya fruit.
2. Aspartyl protease of HIV and protease inhibitor as AIDS drug

Human immunodeficiency virus (HIV) and other retroviruses contain an unfused dimeric
aspartyl protease. The central feature of the active sites is a pair of aspartic acid residues
that act together to allow a water molecule to attack the peptide bond.

Electron push and pull model:




Crixivan, an HIV protease inhibitor, is used in the treatment of AIDS. This protease
cleaves multidomain viral proteins into their active forms; blocking this process prevents
the virus from being infectious.


HIV Protease-Crixivan Complex




     The flaps close down on the binding pocket after substrate has been bound.
Mechanism of specific inhibition of HIV protease




Crixivan is constructed around an alcohol that mimics the tetrahedral intermediate. The
OH group of the central alcohol interacts with two aspartate residues of the active site, one
in each subunit. In addition, two carbonyl groups of the inhibitor are H-bonded to H2O,
which, in turn, is H-bonded to a peptide NH group in each of the flaps. This interaction of
the inhibitor with water and the enzyme is not possible with cellular aspartyl proteases such as
renin and thus may contribute to the specificity of Crixivan.
  chanism for
     3. Carboxypeptidase A cleaves peptide bond next to C-terminal-COO¯ using Zn++
  rboxypeptidase A       (an exopeptidase)




sm for
  all 2001


                    st
                                  Chapter 22                                     30



     1 Step: molecular recognition by Tyr 248 and Arg 145, 127; hydrophobic pocket
            H O Nu A
peptidaseenhanced by Glu 270 general-base cat. & Zn cat. (3 aa coord.)
                             2
                                                                                      2+




 ng
  ged
 t on
 ine                Mechanism for
                    Carboxypeptidase A
                        second step,
                 In the 22
                 Chapter                                                 31

                 Glu 270 functions
                 as a general-acid
                 catalyst increasing
                 the leaving
                 tendency of the
                 amino group

                 Note only few of the proteinases work as amino- or carboxypeptidases, while most are endopeptidases
                  © Prentice Hall 2001           Chapter 22                           35
                 preferably cleaving peptide bonds within a polypeptide chain rather than at its ends.
4. More Zn++ ion catalysis and synthetic model of carbonic anhydrase

CO2, a major end product of aerobic metabolism, is released into blood and transported to
the lungs for exhalation. It is hydrated in the blood to carbonic acid, and dissociates H+
(pKa = 3.5) to bicarbonate.

This hydration proceeds at 0.15 s-1; the reverse 50 s-1, hence K = 5.4 × 10-5, giving a ratio
of [CO2] to [H2CO3] of 340:1.

Carbonic anhydrases accelerate CO2 hydration dramatically. The human carbonic
anhydrase II can hydrate CO2 at ~106 s-1. This is required because CO2 hydration and
HCO3¯ dehydration are often coupled to rapid processes like transport processes or eye
secretions.
Human carbonic anhydrase II and Its Zinc Site. (Left) The zinc is bound to the imidazole
rings of three histidine residues as well as to a water molecule. (Right) The location of the
zinc site in the enzyme.




 Mechanism of action




Histidine Proton Shuttle. (1) His 64 abstracts H+ from Zn++-HOH, generating a
nucleophilic ¯OH and His-H+. (2) The buffer (B) removes a proton from His-H+,
regenerating the unprotonated form.
Synthetic model accelerates hydration of carbon dioxide more than 100-fold




                              Step 1, pKa 7
                                              Mechanism for Lysozyme
  5. Lysozyme hydrolyzes bacterial cell wall composed of -glycosidic linkages

  In lysozyme, a -glycosidase that degrades the cell walls of some bacteria, the active site is
  made up of residues 35, 52, 62, 63, 101, and 108 in the sequence of the 129 amino acids
                        As Lysozyme binds the substrate, it change
                                              

                        shape slightly, distorting the sugar residue
                        site D, into a half-chair
  Mechanism for Lysozyme destabilizes the reactant, relative to th
                       This

                        transition state




© Prentice Hall 2001       Chapter 22                          54
  Lysozyme hydrolyzes bacterial cell wall   © Prentice Hall 2001           Chapter 22
    Mechanism: Asp 52 electrostatic cat. (salt bridge),
               Glu 35 general-acid cat. Then general-base cat. on HOH


     D-ring distorted into half-chair towards TS conformation, favoring glycosidic cleavage.
     max. activity at pH 5.3 (Asp as anion, Glu as acid, surrounded by nonpolar groups)
6. Isomerization of dihydroxyacetone phosphate (DHAP) to glyceraldehyde 3-phosphate (GAP)
This reaction in glycolysis is rapid and reversible. At equilibrium, 96% of the triose
phosphate is DHAP, but reaction proceeds readily from DHAP to GAP because the
subsequent reactions of glycolysis remove this product. Triose Phosphate Isomerase (TIM)
catalyzes this reaction.




Two notable points about TIM

(1) TIM, a kinetically perfect enzyme, accelerates isomerization by a factor of 1010
compared with that by acetate. The k of rate-limiting step is 2 × 108 M-1 s-1, indicating
almost diffusion-controlled encounter of substrate and enzyme.
(2) TIM suppresses an undesired side reaction, from enediol to methyl glyoxal.




In solution, this elimination is 100 times as fast as isomerization. Hence, TIM must
prevent the enediol from leaving the enzyme. This intermediate is trapped in the active site by the
movement of a loop of 10 residues. This loop serves as a lid on the active site, shutting it when the
enediol is present and reopening it when isomerization is completed. The undesirable alternative reaction
is suppressed by having the phosphate group anchored.
Structure of Triose Phosphate Isomerase. This enzyme consists of a central core of eight parallel 
strands (orange) surrounded by eight  helices (blue). This structural motif, called an  barrel, is also
found in the glycolytic enzymes aldolase, enolase, and pyruvate kinase. His 95 and glu 165, essential
components of the active site of triose phosphate isomerase, are located in the barrel. A
loop (red) closes off the active site on substrate binding.
7. Bacterial transpeptidase.
   Penicillin inactivates an enzyme-serine in bacterial cell-wall synthesis

Bacterial cell walls are unique in containing D amino acids, which form cross-links of different
peptidoglycan strands catalyzed by glycopeptide transpeptidase.. The amino group at one end of a
pentaglycine chain attacks the peptide bond between two D-alanine residues in another peptide unit.




                           Sugars are shown in yellow, tetrapeptides in red, and pentaglycine bridges in
blue. The cell wall is a single, enormous, bag-shaped macromolecule because of extensive cross-linking.

Penicillin inhibits the cross-linking transpeptidase disguised as a Trojan horse. The transpeptidase
normally forms an acyl intermediate leading to cross-link formation.




Penicillin gets into the active site of the transpeptidase because it mimics D-Ala-D-Ala:




Penicillin, with a highly strained and reactive 4-mem -lactam, reacts with the transpeptidase to
form an inactive complex, which is indefinitely stable.
III. Enzyme-coenzyme chemistry of electron transfer: NAD+ and FAD

1. Coenzyme and vitamins for electron transfer and structural link

The enzyme function for H¯ transfer requires an electron carrier as coenzyme. The 20 amino acids of
protein are ill-equipped to handle H¯ which is too basic and unstable. Coenzymes are small organic
molecules derived from vitamins. Since H¯ denotes an electron lone pair and H+, H¯ transfer is also lone
pair transfer and hence a redox reaction.


 Coenzymes required in enzyme functions and the corresponding vitamins

Vitamin          Coenzyme                      Typical enzyme     Consequences of deficiency
                                               function

Nicotinic acid Nicotinamide adenine            Oxidation-         Pellagra (dermatitis, depression,
(niacin)        dinucleotide (NAD+)            reduction          diarrhea)
Riboflavin (B2) Flavin adenine dinucleotide    Oxidation-         Cheliosis and angular stomatitus
                (FAD)                          reduction          (lesions of the mouth), dermatitis

These are water-soluble vitamins.

Vitamins are needed in small amounts in the diets of higher animals that lost the capacity to synthesize
them in the course of evolution. The biosynthetic pathways for vitamins can be complex; thus, it is
biologically more efficient to ingest vitamins than to synthesize the enzymes required to construct them.

Note
Vitamin C, a well-known antioxidant of its own right, is not a coenzyme. In the synthesis of 4-
hydroxyproline, an amino acid of the connective tissue collagen, Vitamin C is required for the continuing
activity of the reaction enzyme prolyl hydroxylase. This is a dioxygenase with a tightly bound Fe2+ to
activate O2. The result is Fe3+ in the inactivated enzyme which is reduced by ascorbate to Fe2+.
 Structural link: Adenosine Diphosphate (ADP) is a fundamental building block in NADH, FAD,
coenzyme A, and ATP.




     The adenine is shown in blue, ribose in red, and diphosphate in yellow.

Adenine C5H5N5, a pentamer of HCN, and ribose have prebiotic origin. These ancient
structures are conserved for molecular recognition in various metabolic functions.
             oxidations, the NAD+/NADH ratio is high ~1000
            Anabolic reactions are predominantly reductions -
             NADP+/NADPH ratio is low ~ 0.01
Niacin: The Vitamin Needed fo
           2. Structures of NAD/NADH & niacin vitamin for redox reactions


Redox
            © Prentice Hall 2001                    Chapter 23                          5




       Niacin: The Vitamin Needed for
       Redox
      Niacin: The Vitamin Needed for
      Redox
             Much of the structural complexity of these
 tice Hall 2001                       Chapter 23
          Much of the structural complexity of these recognition
              coenzymes is for molecular
           coenzymes is for molecular recognition
       
            The business end of NAD+/NADP+ is the
           The business end of NAD+/NADP+ is the
              pyridine ring
           pyridine ring




    © Prentice Hall 2001             Chapter 23                             6
           Niacin: The Vitamin Needed for
           Redox
      © Prentice Hall 2001 Chapter 23                                                         6
           NADPH is used almost exclusively for reductive biosyntheses (anabolism), whereas
          The NAD +/NADH couple is (catabolism). The phosphoryl group on
           NADH is used primarily for the generation of ATP used almost
           exclusively in catabolic reaction whereas the
           NADP tag that enables couple is used in anabolic
           NADPH is a+/NADPHenzymes to distinguish between when to use what.
           reactions
          Since catabolic reactions are predominantly
           oxidations, the NAD+/NADH ratio is high ~1000
          Anabolic reactions are predominantly reductions -
           NADP+/NADPH ratio is low ~ 0.01



           © Prentice Hall 2001                    Chapter 23                           5
3. Glyceraldehyde 3-Phosphate dehydrogenase and NAD+

  NAD+-binding region in dehydrogenases. In glycolysis and the subsequent conversion of pyruvate,
the three dehydrogenases (glyceraldehyde 3-phosphate dehydrogenase, alcohol dehydrogenase, and
lactate dehydrogenase) have in common a domain for NAD+ binding. This nucleotide-binding region is
made up of 4 helices and a sheet of 6 parallel strands, often called a Rossmann fold. This fold is likely
a primordial dinucleotide-binding domain that recurs in the dehydrogenases.

The nicotinamide-binding half (yellow) is structurally similar to the adenine-binding half (red). The
NAD+ molecule binds in an extended conformation.




 Structure of Glyceraldehyde 3-Phosphate Dehydrogenase. The active site includes a cysteine
residue and a histidine residue adjacent to a bound NAD+.
 Catalytic mechanism of glyceraldehyde 3-phosphate dehydrogenase. The reaction proceeds through
a thioester intermediate, which allows the oxidation of glyceraldehyde to be coupled to the
phosphorylation of 3-phosphoglycerate.




Why are the two reactions coupled?

Advantages are seen by comparing with the activation energies when oxidation and acyl
phosphate formation are uncoupled.
 Free-Energy Profiles for Glyceraldehyde Oxidation Followed by Acyl-Phosphate Formation.

(A) A hypothetical case with no coupling between the two processes. The second step
must have a large activation barrier, making the reaction very slow. (B) The actual case
with the two reactions coupled through a thioester intermediate.
       Flavin Adenine Dinucleotide and
       Flavin Mononucleotide
4. Flavin redox coenzymes FAD/FADH2 are covalently bound to enzyme and complimentary to
   NAD/NADH

Mechanism of dehydrogenation



   Flavin Adenine Dinucleotide and
   Flavin Mononucleotide
 There are multi e-addition sites in FAD: S attacks C4a, C at N5 as shown for oxidation
                                            ¯               ¯
of amino acid to an imine (net removal of 2 H•)
          Enzymes that use oxidizing coenzymes
           other than NAD+ may still require NAD+
           to reoxidize the reduced coenzyme

    © Prentice Hall 2001                 Chapter 23                                  12




 © Prentice Hall 2001                     Chapter 23                                         11


Why FAD as another electron carrier and why not use NAD+ throughout

 Along this line, would it not be even simpler to use molecular oxygen. In the citric acid
cycle, O2 does not participate directly. True, the cycle operates only under aerobic
conditions because NAD+ and FAD can be regenerated only by electron transfer to O2.
 Look at succinate oxidized to fumarate by succinate dehydrogenase. The hydrogen
acceptor is FAD rather than NAD+, which is used in the other three oxidation reactions in
the cycle.


 In succinate dehydrogenase, a histidine side chain is covalently attached to C-8 methyl of
the tricyclic ring of FAD (denoted E-FAD).
 FAD is the hydrogen acceptor because the free-energy change is insufficient to reduce
NAD+. FAD is nearly always the electron acceptor in oxidations that remove two
hydrogen atoms from a substrate.
 Such difference is also seen in the generation of ATP (e-transport chain in the cycle
yields a proton-motive force):
                  ~ 2.5 ATP per NADH, and 1.5 ATP per FADH2.
 Succinate dehydrogenase contains iron-sulfur clusters, 2Fe-2S, 3Fe-4S, and 4Fe-4S. FADH2 produced
by the oxidation of succinate does not dissociate from the enzyme, in contrast with NADH produced in
other redox reactions. Rather, 2 electrons are transferred from FADH2 directly to iron-sulfur clusters of
the enzyme and ultimately to O2.
FAD precedes NAD+ in major metabolic pathways

Below shows major metabolic pathways with similar sequences of reactions in common:
oxidation, hydration, and another oxidation, resulting in  methylene group (CH2)
converted into a carbonyl group (C=O).




Oxaloacetate is regenerated for another round of the cycle, and more energy is extracted in
the form of FADH2 and NADH.
IV. Enzyme-coenzyme chemistry of group transfer: Imine chemistry of pyridoxal and cryptic carbanion
    chemistry of thiamin

1. Coenzyme and vitamins for group transfer

Vitamin       Coenzyme              Typical enzyme function Consequences of deficiency

Pyridoxine    Pyridoxal             Group transfer to or from Depression, confusion, convulsions
(B6)          phosphate             amino acids
Thiamine      Thiamin               Aldehyde transfer         Beriberi (weight loss, heart problems,
(B1)          pyrophosphate                                   neurological dysfunction)

Other examples of group transfer not discussed here

Biotin Biotin-lysine complexes      ATP-dependent carboxylation and      Rash about the eyebrows, muscle
       (biocytin)                   carboxyl-group transfer              pain, fatigue (rare)
Folic Tetrahydrofolate              Transfer of one-carbon components;   Anemia, neural-tube defects in
acid                                thymine synthesis                    development
B12    5 -Deoxyadenosyl             Transfer of methyl groups;           Anemia, pernicious anemia,
       cobalamin                    intramolecular rearrangements        methylmalonic acidosis

These are water-soluble vitamins.
                              Pyridoxal Phosphate
                                  In each of these reactions, one of the
                                   bonds to the -carbon of the amino acid
        2. Pyridoxal in group transfer reactions
                                   is broken in the first step




  Pyridoxal Phosphate       © Prentice Hall 2001           Chapter 23




        Structures of pyridoxal (free and enzyme bound) and reactivity
                                                                                          28




        The most important functional group on PLP is the aldehyde. It allows PLP to form
        covalent Schiff-base with a specific lysine -amino group of the enzyme or with the amino
        acid substrates. These Schiff-base linkages are often protonated, with the positive charge
        stabilized by interaction with the negatively charged phenolate group of PLP.




© Prentice Hall 2001                               Chapter 23                                        26
          Pyridoxal mediate group transfer in




       Pyridoxal Phosphate


   © Prentice 2001
© Prentice HallHall 2001                   Chapter 2323
                                             Chapter           26
                                                                26




Pyridoxal Phosphate
            H transfer makes racemization possible



    © Prentice Hall 2001                    Chapter 23         27




rentice Hall 2001                    Chapter 23           27
Mechanism via imine-enamine intermediates: transamination, decarboxylation, transfer of RCHO




    Pyridoxal Phosphate                                        Glutamate (repeat steps above)
          Pyridoxal Phosphate
 Transfer of RCHOH as RCHO




 TransferofH makes L-amino acids




       © Prentice Hall 2001                Chapter 23                                 31




In a transaminase active site, addition of H+ from the lysine residue to the bottom face of
the quinonoid intermediate determines the L configuration of the amino acid product. The
conserved arginine residue interacts with the -carboxylate group and helps establish the
appropriate geometry.
Seleative bond cleavage by stereoelectronic control of PLP Enzymes

PLP enzymes labilize one of three bonds at the -carbon of an amino acid substrate. For
example, bond a is labilized by aminotransferases, bond b by decarboxylases, and bond c
by aldolases (such as threonine aldolases).




How done?

An important principle is that the bond being broken must be perpendicular to the  orbitals of the
electron sink. An aminotransferase achieves this goal by binding the amino acid substrate so that the C-
H bond is perpendicular to the PLP ring.

e.g., Aspartate Aminotransferase. The active site has a PLP Schiff-base linkage with lysine 268. An
arginine helps orient substrates by binding to their -carboxylate groups.




In aspartate aminotransferase, the C-H bond is most nearly perpendicular to the -orbital
system and is cleaved.
In serine hydroxymethyltransferase, the N-C bond is rotated so that the C-C bond is
most nearly perpendicular to the plane of the PLP ring, favoring its cleavage.
           Thiamine Pyrophosphate
          3. Cryptic carbanion chemistry of thiamin
          Structure of thiamin and unique ylidene as carbanion




 Thiamine Pyrophosphate
hiamine Pyrophosphate
  The hydrogen bonded to imine
The hydrogen bonded to thethe imine
   carbon is relatively acidic, pK = 12.7
carbon is relatively acidic, pKa = a12.7




                                                                      pKa ~13
        © Prentice Hall 2001                             Chapter 23                                           13

          Significant role of thiamin in mediating between glycolysis and the citric acid cycle
           Pyruvate produced by glycolysis is converted into acetyl CoA, the fuel of the citric acid cycle.

e Hall 2001
Prentice Hall 2001                      Chapter 23
                                  Chapter 23                                           14     14
Pyruvate dehydrogenase complex of E. coli joins with thiamin for crucial link

Enzyme                         Abbrevn   # chains Prosthetic group Reaction catalyzed
Pyruvate dehydrogenase           E1         24    TPP              Oxidative decarboxylation     of pyruvate
component
Dihydrolipoyl transacetylase     E2        24    Lipoamide      Transfer of the acetyl group to CoA
Dihydrolipoyl dehydrogenase      E3        12    FAD            Regeneration of the oxidized form of lipoamide


Schematic Representation of the Pyruvate Dehydrogenase Complex
The transacetylase core (E2) is shown in red, the pyruvate dehydrogenase component (E1) in yellow, and
the dihydrolipoyl dehydrogenase (E3) in green.




The conversion of pyruvate into acetyl CoA consists of three steps: decarboxylation,
oxidation, and transfer of the resultant acetyl group to CoA.




Pyruvate is decarboxylated at the active site of E1, forming the substituted TPP
intermediate, and CO2 leaves as the first product. This active site lies within the E1
complex, connected to the enzyme surface by a 20-Å-long hydrophobic channel.

Note these steps are coupled to preserve the free energy derived from the decarboxylation
step to drive the formation of NADH and acetyl CoA.
          Thiamine Pyrophosphate
     Mechanism via thiamine ylidene intermediate in decarboxylation & transacetylation


                                      Thiamine Pyrophosphate
                        The resulting intermediate easily can
                                      
  Thiamine Pyrophosphate decarboxylation as electrons
                        undergo
   The ylide carbanion left behind can be delocalized onto the
                        attacks the ketone
    group of an -keto acid (pyruvate)
                        positively charged nitrogen



                       Thiamine Pyrophosphate
       © Prentice Hall 2001            Chapter 23                     15
© Prentice Hall 2001              Chapter 23                     16

                                  © Prentice Hall 2001                     Chapter 23         17




                   © Prentice Hall 2001                  Chapter 23                      20
Schematic diagram of the dimer interface environment around the catalytic center.
Bonding interactions between cofactors and protein are indicated by dashed lines. Residues numbered
>360 are in the  domain of one monomer, while those <187 are in the  domain of the 'other" monomer.
Illustration of complete reaction schemes of the Pyruvate Dehydrogenase Complex

At the top (center), the enzyme (represented by a yellow, a blue, and two red spheres) is
unmodified and ready for a catalytic cycle. (1) Pyruvate is decarboxylated to form the
hydroxyethyl TPP. (2) The dihydrolipoyl arm of E2 moves into the active site of E1. (3)
E1 catalyzes the transfer of the two-carbon group to the dihydrolipoyl group to form the
acetyl-lipoyl complex. (4) E2 catalyzes the transfer of the acetyl moiety to CoA to form
the product acetyl CoA. The disulfhydryl lipoyl arm then swings to the active site of E3.
E3 catalyzes (5) the reduction of the lipoic acid and (6) the transfer of the protons and
electrons to NAD+ to complete the reaction cycle.
Detailed picture of transketolase-thiamine-substrate

Cleavage of the donor substrate D-xylulose 5-phosphate by yeast transketolase
Fundamental structural questions resolved


J. Phys. Chem. A 2005, 109, 7606-7612

Topological Analysis of the Electron Density in Model Azolium Systems for Thiamin
Structure-Function: Sulfur Is the Electron Sink and Positively Polarized Carbanions Act as
Nucleophiles

Donald B. DuPre´*,† and John L. Wong*,‡
Department of Chemistry, University of Louisville, Louisville, Kentucky 40292
Receivaed: March 22, 2005; In Final Form: June 20, 2005

The mechanism of thiamin diphosphate-dependent enzyme reactions requires two carbanion intermediates 1a
and 1b. Neither has been isolated nor detected, but resonance stabilization is assumed to occur with the
thiazolium quaternary nitrogen being the electron sink. We have questioned the electronic nature of these
carbanion intermediates and, in a broader sense, the role of sulfur in the thiazolium moiety. To address these
issues the theory of atoms in molecules (AIM) was used to acquire quantitative electron distributions in
thiazolium 2, oxazolium 3, and imidazolium 4 as cations and zwitterions. Among the heteroatoms, only sulfur
acts as an electron sink. This is corroborated by a similar behavior in phosphorothioates. Further, the formal
carbanion at C2 and Ca of the intermediates are positively charged and their nucleophilic character is explained
with AIM theory by comparison with the C- of model 5a and C- of model 6a. C2 of 2a excels in lone pair
coverage in the -plane, surpassing the C- in acyclic 5a and other cyclic ylidenes, and hence, is a more
effective nucleophile. The C- of 6a reveals a depletion area centered in the -plane but shows lone-pair
concentration above and below the plane. Unlike 6a, the AIM properties, bond length, and bond order of 2b
indicate no lone-pair on C but essentially a double bond across C2-C. Thus, the nucleophilic behavior at
C of 1b is based on the enamine chemistry induced by an electrophile.

SCHEME 1: Thiamin Diphosphate Intermediates




Contour plot of the Laplacian of the electron density showing regions of charge concentration in a plane perpendicular to the 5-
membered ring and approximately bisecting the N3-C2-S1 bond angle of the thiazolium ylidene 2a.

								
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