ENZYMOLOGY  -  Enzyme from the Greek ένζυμο,  énsymo,
                                     which means én ("in")   and  simo  ("yeast")
enzymes regulate metabolic reaction rates
-  i.e., they control metabolism
            molecules (mostly protein vs ribozymes) that accelerate
            or catalyze chemical reactions (A--->B) in cells by
            breaking old covalent bonds & forming new covalent bonds

a biological catalyst… but, different from a chemical catalyst -
have complex structure (sequence of aa’s) act only upon
            a specific substrate do not change direction (energetics) of rx
catalysis* = acceleration of rate of a chemical reaction by a catalyst
          - enzymes convert substrates to products w/o changing themselves

 ex: é cAMP Protein kinase A[] - group of enzymes that phosphorylate proteins

                                                                                 pages 79-86; 88-92.








  1st enzyme crystallized UREASE [EC], 1926 James Sumner
2 NH2-C0-NH2   +   2 H 2O     ----->     4 NH+4   +    2 CO2 
   Sumner's bioassay = injects rabbits with urease from Jack beans
                         &  the ammonia produced... killed the bunnies

           - to date just over 1,000 enzymes purified & about 100+ crystallized, out of some  105
         - except for ribozymes (that also have catalytic activity) - all are proteins
         - proof that biological activity is due to an enzyme has usually been...
                                  to note the
loss of biological activity as result of
proteolytic digestion
       Some important dates in early
Enzyme History
            1833  Payen & Peroz - alcohol precipitate of barley holds heat labile
(proteins) that converts starch to sugars
             1878  Kuhn - coins term 'enzyme' : Greek "in yeast"
          1897  Hans Eduard Buchner - yeast 'juice'  +  sugars (jelly) = bubbled gas & ETOH
          1898  Ducleaux - uses suffix "ASE" for enzyme naming

   1900  E. Fischer - stereospecificty of enzymes is discovered 






       E + S <---> [ES] <---> E + P

      enzymes catalyze reactions by 
      lowering the
energy of activation... Ea
*            Karp figure


There is no difference in free energy between an enzyme catalyzed reaction and an uncatalyzed react-ion, but an uncatalyzed reaction requires a higher energy input than a catalyzed reaction.


                Catalase:   2H2O2   --->   2 H2O  +  O2 
                      condition            Ea(cal/mol)          Rate (lt/mol/sec)

              no catalyst       18,000              1.0  x  10-7
              Fe catalyst       10,000            56.0
              catalase           2,000              4.0  x  106
        turnover number - number of substrate molecules converted to 
        product per second for a single enzyme molecule     Karp table 3.4 pg 93







Terminology              substrate, product, enzyme... self explanatory
Ribulose biphosphate carboxylase/oxygenase


cofactor: small inorganic ions... mostly metal ions: Cu, Mg, Mn, Fe 
      which act as activators &/or inhibitors of activity

coenzymes : small non-protein ligands that catalyze reactions…
                    +/- electrons,  transfer a group, 
                    form or break a covalent bond    
LIPOIC acid : oxidative de-COOH of alpha-keto acid
NAD+ (NADP+) : dehydrogenation; 
                                    H+ carrier
and/or electron transfer
CoASH : acyl carrier via sulfhydryl (-SH) 
Vitamins : acscorbate, cyanocobalamin, folic acid...     

prosthetic group:  large complex organic molecules, 
                           which may have catalytic activity (heme)







Terminology continued           Lysozyme's active site binds polysaccharide chains
    active site   portion of enzyme which folds to precisely fit (mcb 3.21*3.17*) the contours
                      of a  substrate via weak electrostatic interactions  &  facilitates bond reactivity


    enzyme-substrate complex:   unique joining of enzyme & substrate at active site
       What does an ES Complex do?
            - holds substrate out of aqueous solution
            - holds substrate in specific orientation, close to transition state to allow reaction to occur
            - reduces ability of free rotation & molecular collisions with non-reactive atoms
            - allows amino acid side chains to alter local environment:
                        can change ionic strength, pH, add or remove H-bonds to substrate   
          [Rubisco   &   cAMP-PKA   &    serine proteases*]
                             Analogy:  a nut & bolt held in your hand decrease the Entropy of their
                                            binding over a random mix of nuts and bolts in a toolbox.  










Mechanism of Enzyme Action:     3 examples...
     the chemical reaction scheme by which an enzyme acts upon its substrate

   1.  catalytic action of cAMP dependent Protein Kinase A  mcb3.18*- e's of ATP delocalized by LYS
       & Mg+2; new bond forms between SER-OH &
γP; bond between βP-γP broken = ADP + P-protein.
   2serine protease hydrolysis of peptide bonds mcb3.25a
*. catalytic site holds ser195, asp102,
        & his57:  -OH of ser195 attacks C=O of peptide bond & transition state is held by H-bonds;
        e's break peptide bond & release part of protein; H-O-H is split & other half released.
   3.  another example LYSOZYME :      
               - an enzyme that cuts polysaccharide glycosidic bonds by hydrolysis (adds H2O
  - breaks glycosidic bond (… -C-O-C- …) via bond strain & distortion of glu & asp
               - active site is a long groove, holding six sugar units...
                           has 2 acidic side side chains (
ASP & GLU) hold substrate

enzyme binding of substrate, bends bonds from a stable state,  lowering Ea.
acidic side group of GLU provides [high] of H+  ions for acid hydrolysis,
and negative charged ASP stabilizes + charge
* of the transition state.








 Classification of Enzymes

Enzyme Commission - IUBMB International Union Biochemistry & Molecular Biology
                                        some history of enzyme nomenclature by the IUBMB       

  4 digit Numbering System    []

    1st #...      one of 6 Major Classes of Enzyme Activity

    2nd #...     a subclass (type of bond acted upon)

    3rd #...     a subclass (group acted upon, cofactor required, etc...)

    4th #...      a serial number… (order in which enzyme was added to list)








1. Oxidoreductases...
[ dehydrogenases]
             catalyzes oxidation reduction reactions, often using coenzyme as
                             Alcohol dehydrogenase   [EC]  ethanol + NAD+ ---> acetaldehyde + NADH
   2. Transferases...  catalyze the transfer of functional group         
                               Hexokinase   [EC]   D-glu + ATP ---> D-glu-6-P + ADP
   3. Hydrolyases… catalyzes hydrolytic reactions adds water across C-C bonds 
                             Carboxypeptidase A   [EC  [aa-aa] n + H2O ---> [aa-aa] n-1 + aa
   4. Lyases...  cleave C-C, C-O, C-N & other bonds often genrating a C=C bond or ring
                               Pyruvate decarboxylase   [EC   pyruvate ---> acetaldehyde + CO
   5. Isomerases [mutases]  catalyze isomerizations
                              Maleate isomerase   [EC]  maleate ---> fumarate 
   6. Ligases…   condensation of 2 substrates with splitting of ATP
                              Pyruvate Carboxylase   [EC  PYR + CO 2 + ATP ---> OAA + ADP + P
                some common functional classes of enzymes***











Enzyme Kinetics…
    defines the physical & chemical properties of enzyme by mathematical and/or graphical
     expression of the
reaction rates of enzyme catalyzed reactions

2 H2O2   ---->   2 H2O   +   O2

   Characteristic Enzyme Kinetic Curves:
                    or     how to determine if the reaction   A —> B   is enzymatic 

Observed Enzyme Kinetic Reaction Curves :
   1.    Rate (0.8 ml O2/min)   Vs.   [ E ]      a classical 1st order linear plot
   2.    Rate   Vs.   pH
   3.    Rate   Vs.   Temperature    figure*
   4.    Rate   Vs.   [S]        most CHARACTERISTIC curve… a plot of  v  vs.  [S]*
                            rate of O2 production*        








v vs. [S] curve defines a rectangular hyperbola
at low [S], rate is directly proportional to [S]
at higher [S], rate declines giving a rectangular hyperbola

How to gather data points for enzymes curve
                            - ecb 3.27*


1st & 2nd order reaction kinetics are NOT sufficient 
                                                       to describe the rectangular hyperbola of enzyme reactions   
     A       <--k1-->  B        for 1st order Rx           dP/dt    =     k1 [A]        linear response 
     A + B  <--k2-->  C       for 2nd order Rx          dP/dt    =     k2 [A] [B]   also linear













    in 1913 Leonor MICHAELIS  &  Maud MENTEN Kinetics 
                                   proposed a mathematical modeling of enzyme reactions 
                                         i.e., algebraic expressions to define a rectangular hyperbola

    k1                  k3   
E + S <---------> ES <--------> E + P
 k2                 k4

          M&M assumptions                                                
                 1)  rate formation ES complex from E + P is negligible   i.e., can ignore the rate constant k4
2)  rate LIMITING step is disassociation of   ES  to  E + P   =   k3  
           3)  most important state of the ENZYME is termed FREE ENZYME 
         free enzyme            =     Et - ES   
                                bound enzyme        
 =     ES 

  total enzyme            =     Et = [Et - ES] + [ES]


         "Die Kinetik der Invertinwirkung" Michaelis, L.; and Menten, M.L. (1913) Biochem. Z. 49, 333-369.




Derivation of Michaelis-Menten Enzyme Kinetics...
   the derivation of equation occurs at a time when...
rate of formation of ES complex is equal to rate of destruction (break down),
             i.e., at equilibrium, when
[S] >>>> [E] so that total E is bound in ES complex and
             reaction works like a 1st order reaction enzyme catalyzed reaction

   the rate limiting equation thus becomes         v = (dP/dt) = k3 [ES]
   it would be easy if we could measure the concentration of [ES]...  say in a spectrophotometer
     but, its presence is fleeting.... 
so then the real purpose of the derivation of M&M kinetics 
     is to be able to express 
[ES]  in terms of  E  &  S  alone, which are measurable
measuring [ES]...  is very difficult    [ stopped flow apparatus ecb 3.28* ]
the M & M equation is then :       v  =   Vmax [S]
                                                                                       Km + [S]
          FOLLOW ALONG         LINK to pdf copy for printing...  





Derivation of Michaelis-Menten Equation
 k1                     k3
E + S   1      ES      1   E + P
k2                    k4

  rate limiting step is       DP/Dt   =  k3  [ES]                    (&   DP/Dt  =  v )

1.  Rate of formation of ES complex     DES /Dt   =   k1  [E T - ES]  [S]

2.   Rate of destruction ES complex      DES /Dt   =   (k2 + k3)  [ES] 

3.   Steady State Equilibrium        k 1  [ET- ES] [S]  =  (k2+ k3) [ES] 

4.   Michaelis Constant  (Km)                 ( k2+ k3)    =      [ET - ES]  [S]
( k1)                        [ES]

              down                              Km    =    ( k2+ k3)   =       [ET - ES]  [S]
( k1)                        [ES]   

5.   Solve for  [ES ]                           [ES]   =         [ET]    [S]   
                                                                               (Km) + [S]

6.   Substitute in above    DP/Dt  =  k3 [ES]           v     =        k3   [ET]    [S]   
                                                                                                (Km) + [S]  

7.   Substitute Vmax for  k3 [ET]                            v    =      Vmax    [S] 
                                                                                       Km  +  [S]    








     Km - the Michaelis Constant
                ...is "inherent tendency" of reactants to interact chemically for speed of rx
                ...is a constant that is independent of [S] or [E]
                ...is a mathematical interpretation of an enzyme action 
                ...is the
substrate concentration...  when enzyme velocity is equal to ˝ Vmax

                               thus, when  =   ˝ Vmax                  Vmax    =       Vmax      [S]  
                                                                              2              (Km) + [S]

             Solve  1  =           [S]              & rearrange
                        2         (K m) + [S]           Km + [S]  =   2   [S]     thus  Km  =  [S]

                 native values for Km's  10-1  to 10-7 M        average Km is  10-4 M   






 Km is a characteristic physical property for each and every different enzyme.

                                it is independent of [
E]  and is independent of [S]
                                it measures "
realtive afffinity" of an enzyme for its substrate


    suppose there's more than 1 substrate for an enzyme [phosphatases], then each has its own Km
ex:  one enzyme with 2 substrates each with following Km's -  1 mg   &   25 mg
         thus, one takes less substrate to reach same rate…  ˝
Vmax rate        figure*

                     many enzymes have individual steps in a complex reaction sequences, 
                     each step has its own Km's…
Km is a complex function of many individual rate constants

not all enzymes are treatable by M & M kinetics…
regulatory enzymes (multi-subunits) are not treatable by M&M kinetics.





  Some ways to determine Km...
       1by extrapolation from a graph of a M & M standard curve      v  vs.  [S]               











 2.  by transformation of M & M curve graphically
     Hans LINEWEAVER & Dean BURK plots               Karp fig 3.18     ECB fig 3.27*
       take the reciprocal of both sides of M&M equation & plot
         1   =   Km      x    1   +    
         v     Vmax          S      Vmax        x intercept equals 
    y intercept equals   1/Vmax

       EADIE - HOFSTEE Plots* 
v     vs.     v/[S]                                 slope equals  Km
   y intercept equals  Vmax














 Eadie Hofstee Plot

The Eadie-Hofstee plot is a way of plotting kinetic enzyme data so as to yield a straight line for reactions obeying Michaelis-Menten kinetics. This is done by plotting reaction velocity (V) versus velocity/substrate concentration (V/[S]). The slope of the line is equal to -KM and the y-intercept is Vmax.


An advantage of an Eadie-Hofstee plot over a Lineweaver Burk plot (which plots 1/V versus 1/S) is that the Eadie-Hofstee plot does not require a long extrapolation to calculate Km. 

                                      MORE SAMPLES of GRAPHICAL PLOTS





Lineweaver-Burk Eadie-Hofstee








 Definitions of Enzyme Activity:
                             a way of expressing the physical properties of an enzyme...
                             most often measured by relative rate of  substrate ---> product
          1 unit ACTIVITY that amount enzyme protein which converts
    1 umole substrate per min at 25oC & optimal pH 
          1 unit SPECIFIC ACTIVITY
    # units per mg of protein present   (e.g., 37umole/min/mg protein)
          1 unit MOLECULAR ACTIVITY
    # units per umole of purified enzyme       (e.g., 12 units/umole enzyme)









Enzyme Inhibition...  reducing reaction rates via binding of non-substrate molecule

   2 classes of inhibitors: 

1. IRREVERSIBLE - inhibitor molecule can not be easily removed from enzyme
         thereby reducing the number of working enzyme molecules.  
         i.e, enzyme is physically altered by binding of inhibitor - reducing its amount
alkylating agents like iodoacetamide (bind to -SH’s)
organophosphorous compounds- nerve gases (bind to SER)
antibiotic drugs, such as penicillin
form covalent link to active site

2. REVERSIBLE - enzyme activity may be restored by removing the inhibitor 
           and are thus treatable by M & M kinetics
                 2 major types of reversible inhibitions... 
                      a.   COMPETITIVE 
              b.  NON-COMPETITIVE












inhibitor binds to E & forms an [EI] complex at the active site     ecb 3.29*

inhibitor often looks like substrate... fools active site & binds

extent of inhibition is concentration dependent [inhibitor is at fixed conc]

                          i.e., it can be overcome if [S] is very high,   i.e., [S] >>> [I]

                one classical example is malonic acid inhibition of SDH

                easy to demonstrate is via Lineweaver-Burk plots*

                            µ        shows Vmax is SAME,   but  Km  value is increased









inhibitor binds to E, forms an [EI] complex not at the active site

inhibitor often bears no structural relationship to substrate

removes a net amount of active enzyme, i.e., lowers total [E]

                        i.e., it can NOT be overcome, even if [S] is very high

              easy to demonstrate via Lineweaver-Burk plots

µ  shows Km is SAME  &   Vmax is different     figure*







 Some Examples of Native  of Enzyme Inhibition:


1. Irreversible Enzyme Inhibition & Mechanism of Action of Some Antibiotics...

     Antibiotic - a natural molecule (often made by bacterial cells) that can kill other
                        bacterial cells (& without hurting eukaryotic cells:
they're insensitive)

     Penicillin - any one of a group of antibiotics derived from the fungus Penicillium. The action
        of natural penicillin was first observed in 1928 by British bacteriologist Alexander Fleming,
        and recognized as anti-bacterial by Howard Florey and others.

             Penicillin is a substrate-like molecule* similar to bacterial peptidoglycans, which
                              naturally cross-link in the bacterial cell walls & favor rigidity.
             penicillin  works by IRREVERSIBLE binding to active site of enzymes that link
                              peptidoglycans; forms covalent link, removing enzyme, reducing
                              its Vmax; weakens bacterial walls eventually rupture & cells die.







 2a.  Competitive Enzyme Inhibition  and  Mechanism of Drug Action

         ACE Inhibitors - drugs that bind to the enzyme's active site & reduces its activity

           ACE - Angiotensin Converting Enzyme: a proteolytic enzyme that cuts Angiotensin I
                         a polypeptide of 10 amino acids to Angiotensis II (of 8 amino acids).     figure*

           Angiotensin II promotes hypertension ( high blood pressure - HBP ) via vasoconstriction

                       in 1960's John Vane discovered TEPROTIDE in Brazilian pit viper venoms, a
                       nonapeptide (9aa =
Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro) which functions as
ACE competitive inhibitor, by binding to the active site of the ACE enzyme.

              today there are a number of synthetic peptide ACE inhibitors, all called "prils"...
                                              (lisinopril, captopril, trandolapril
, moexipril, ramipril, etc...) 

              another competitive inhibitor example using Viagra*







  Mechanisms of Protein & ENZYME RATE Regulation…

   5 approaches commonly employed by cells...


1.  by controlling number of enzyme molecules present (gene action)

2.  by sequestering (compartmentalizing) – for example into lysosomes, mitochondria

      3.  by proteolytic clevage - converting inactive peptides to active enzymes
                - often involves hormones and digestive proteases
                - pancreas makes zymogens...   (an inactive enzyme large precursor)
                                                               ex: trypsionogen & chymotrypsinogen
                - enterokinase, an aminopeptidase from the lining of small intestine
                            it hydrolyzes trypsinogen to trypsin (active form), which itself
                            hydrolyzes chymotrypsinogen in chymotrypsin





4.  by adjusting reaction rates of existing enzyme (ala... M&M kinetics)

STIOCHIOMETRIC controls - limit amount substrate present

ALLOSTERY -  [allosteric kinetics]
                      - binding of a ligand results in a
change of 3o/4o conformations
common in multimeric proteins/enzyme complexes
                      - allosteric proteins have 2 binding sites: 
active site = substrate
allosteric site = regulator ligand
                            aspartate transcarbamylase initial enzyme in pyrimidine synthesis- ATCase*
active form =
+ catalysis  & inactive conformation = - catalysis
ligand often serves as substrate, activator, or inhibitor (or all three)






   Examples of Ligand induced Allostery...

          a)  Cooperative Binding
: binding of one ligand affects subsequent bindings
+  =  enhanced subsequent bindings
-  =  sequential binding is inhibited
HEMOGLOBIN:  binding of 1 O2 to a heme = Δ-local conformation
lowers Km of binding of additional O2 to other
                                          subunits  (mcb3.30
*) chains

          b)  Ligand-induced activation of catalysis: (ex - PKA)
                1. inactive PKA is activated by cAMP...
                      binding of cAMP induces 
Δ-conformation, so that a tetramer dissociates
                      into 2 active monomers & a dimeric regulatory subunit    (mcb15.23a*)
                               thus a hormone signals --> cAMP --> active PKA dimer     (mcb15.23b)
                               without PKA we have an inactive tetramer

               2. GroEL chaperonin:  is made of 2 multi-subunit rings            (mcb3.17*)
                      binding of ATP & GroES to GroEL results in a tight peptide binding complex,
                      which opens the folding cavity allowing efficient folding of nascent proteins




           3. Calmodulin*: a Ca binding protein often in monomeric units that changes shape
                      when bind Ca. Calmodulin, a helix-loop-helix protein, has 4 Ca binding sites...
                      4 Ca ions bind = 
Δ-conformation - now binds target proteins = now switched on
4. GTPase super family:  a group of allosteric plasma membrane proteins
                            switching between active/inactive, includes Ras & G-proteins
                     GTP Binding Proteins (G Proteins)
  are Active  when  GTP  is bound to protein                                  mcb 3.32*
Inactive  when  GTP  is hydrolyzed to GDP
serve as molecular switches, esp. cell signaling - LG later

involves COVALENT MODIFICATION of existing enzyme...
                      - addition of
P to an inactive enzyme --> activate enzyme via P transfer
                                               [reversible phosphorylation changes protein conformation] 

              - done by -
PROTEIN KINASES,  which transfer P from ATP       mcb 3.33*
                                             tyrosine kinases add P to TYR residues of enzymes de-activating them
                                        serine/theronine kinases add P to SER or THR residues
                                      - PROTEIN PHOSPHATASES...  dephosphorylate, thus inactivating





   Net RESULTS of Protein Regulatory mechanisms... all Help Control Metabolism.

        feedback inhibition (negative allosteric regulation
                                an initial enzyme is inhibited by end product

        prevalent in the amino acid biosynthetic pathways - figure*

        balance of inhibition & stimulation (glycogen metabolism via cAMP)
                                epinephrine stimulated increase in cAMP, which activates PKA
                                cAMP involved in glycogen to G-1-P conversion:
                                    a. inhibits glycogen synthesis
                                    b. stimulates glycogen degradation   (mcb15.25*)  


   µ  Primary mechanism of action is altering enzyme's activity  (negative or positive*)


     next lecture                                                               protein & enzyme practice questions
























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