HIV peptidase


- a Species Specific polymer of amino acids with biological activity  

  lysozyme - hydrolyzes peptidoglycans in bacterial cell walls    next                             

Proteins are Classified by Function







Enzymes  catalytic activity    A ------> B
Transport Proteins bind & carry ligand molecules (hemoglobins)
Storage Proteins ovalbumin (egg), ferretin (iron), casein (milk)
Contractile Proteins can contract, change shape (actin & myosins) and
  make up elements of cytoskeleton & muscles
Structural Proteins provide support... collagen fibers of tendons (wounds),
  elastin of ligaments, keratin of hair & feathers,
  fibroin of silk & spider webs
Defensive Proteins provide protection:  antibodies (IgG),    fibrinogen,
    thrombin,  and  snake venoms
(digestive enzymes)
Regulatory Proteins regulate metabolic processes: includes hormones,
  transcription factors & enhancers
  nomeclature         protein nomenclature is based on solubility*

      table 1*

        Concept Activity - 5.4 - Protein Functions







 Structure and Properties of Proteins
  PROTEINS - are  POLYMERs  of amino acids w biological reactivity 
                                                     made of alpha - amino acids (
commonly 20)
    in humans only 11 of 20 amino acids can be synthesized; 9 others (essential aa's) must be eaten.
                                                                                                 [HIS, ILE, LEU, LYS, MET, PHE, THR, TRP, & VAL]
    STRUCTURE of Amino Acids               fig of alpha amino acid*
   aa's contain 2 functional groups: a carboxyl group (-COOH)  &   an amino group (-NH2)
        & each of these functional groups are often ionized* at physiological pH   

    ZWITTERION... 2 functional groups of opposite charges in same molecule (as in aa's)
                       Isoelectric Point - pH where there is no net charge in molecule
                       pK - pH at which all the groups are 50% ionized & 50% non-ionized  
                     What is charge of glycine at pH 3.0 ?        at pH 9.0 ?      (ans*)








NH2-groups are bound to an asymmetric alpha carbon
                                   β N-C-C-COOH        γ N-C-C-C-COOH
20 different chemical  (R)  groups make the common aa's  &
                         20 ubiquitous & universal amino acids are found in all living systems
                   every amino acid (but one) exists in two isomeric molecular forms:
                                    each with different arrangements of atoms in space   
                    i.e., 2 optical isomers or mirror images of each other   (left-right handed*)    
                                    molecules that exhibit handed-ness are said to be CHIRAL
                                    and the mirror images are referred to enantiomers

                                    but only the L-optical isomer aa's occur in biological proteins
... an anomaly of molecular evolution?




aa's are CLASSED by TYPE via chemical properties of R or Side groups...

Polar Charged
negatively charged amino acids - ASP & GLU
   R group with a 2nd COOH that ionizes* above pH 7.0    
Polar Charged
positively charged amino acids - LYS, ARG, HIS
   R group with a 2nd amide* that protonates below pH 7.0   
   POLAR      UNCHARGED includes SER, THR, TYR, ASN, GLN    (cys)                   
   are soluble in water, i.e., hydrophilic
  (attract H-bonds)                figure*
   contain hydroxyl or amino functional groups    
includes GLY, ALA, VAL, LEU, ILE, PRO                                  figure*
   all contain only hydrocarbons R groups   =  
AROMATIC (hydrophobic non-polars)    PHE & TRP     (TYR)    &    METCYS
   all contain R groups with    ring structures*         or       Sulfur*   







peptide bond COVALENT LINK between carboxyl end (COOH) of aa-1
             &   amino end (NH2) of aa-2   =   Peptide Bond

forms a dipeptide*   or eventually a polypeptide*        (figure)
linked by a condensation reaction (removes water = dehydration)
         peptide bond is shorter & stronger than C-C
         it's intermediate in length & strength between C-C  &  C=C
         i.e., longer, but weaker than  C=C, but acts like one, thus there is
         no free rotation (attached group in same plane)   figure*

                                                      Concept Activity - 5.4 - Protein Structure






      Examples:    Some Naturally Occurring Oligopeptides (small polypeptides 40 aa or less)
 insulin  2 polypeptides  1.  alpha chain of 30 aas
                          2.  beta chain of 21 aa      regulates blood sugars
 glucagon  pancreatic hormone of 29 aa, opposes insulin action
 corticotropin  39 aa anterior pituitary hormone- stimulates adrenal cortex
 oxytocin  9 aa hormone of posterior pituitary that
        stimulates uterine contractions
 bradykinin  1 aa hormone that inhibits inflammation via smooth muscle contarction
 relasing  factor
3 aa  peptide of hypothalmus that stimulates 
         release of thyrotropin
 enkephalins      CNS peptides that bind to brain cell receptors... 
        have analgesic reaction of pain deadening
 NutraSweet  dipeptide of  L-aspartyl-phenylalanyl methyl ester 3D-fig

                                                                      examples: do not memorize







 FRACTIONATION - Procedures for Isolation & Purification of "new" protein...
  Crude Homogenates  "grind & find"        [ mortar/pestle, blenders & homognizers
  homogenize cells      grinders, 
sonicators,  &  barocycler-(bbi) ]
  Differential Centrifugation  subcell fractionation by centripetal force* in a centrifuge    
 speeds to 250,000xg --> [supernatant & pellet fractions]
             Sumanas, Inc. cell fractionation animation* listen at home
  Column Chromatography - separates proteins from one other via flow thru media in a glass column

    gel filtration

    concept figure 1* [exclusion chromatography]    
    Sephadex - porous carbohydrate polymer beads
                      retard small MW proteins &
                      pass large MW proteins
     figure 2
*   based on SIZE   gel filtration methodology

                        ion exchange

 protein is retarded by binding to ionic charge
      on column's media  (anionic or cation)     figure


 polymeric beads with special ligands* which
      bind specific proteins, but not other proteins    end 7









Gel Electrophoresis    - passage of protein mixture through a porous gel via an
         electrical charge that results in  separation of blood proteins by size & charge
      PAGE   polyacrylamide gel electrophoresis PAGE*       [gel chambers]
 proteomics - protein fingerprints can indicate evolutionary relations


(sodium dodecyl sulphate)- separates proteins via mass (MW)
            1 SDS molecules binds equal to # peptide bonds   
*  &  animation of SDS  electrophoresis*  &  procedures

isoelectric focusing

 proteins migrate thru gels to their isoelectric point,
pH where no net charge & then stop migrating*

2-D electrophoresis

 combines isoelectric focusing & SDS-electrophoresis
   procedures*    and      a sample human leukemic gels*  
isoelectric focusing & SDS electrophoresis databases  
         Identification &
 colorimetric reaction [Biuret & Bradford] where amount of
  color produced is proportional to amount protein present 
                  [ a Standard*Curve
* ]      advertisement







  • Variety of linear amino acid sequences is infinite...  
  • A protein of 100 amino acids made w the 20 different known amino acids
          can have 20100 different linear sequence arrangements [
    1.268 x 10130 proteins]
  • Average protein = 300 to 400 aa's  & has a MW of 30,000 to 45,000d        
  • most often has a globular (spherical) 3-D shape  &  is  negatively charged
  • Fred Sanger - 1958 Nobel prize for determining aa sequence of INSULIN
  • to date some 1,000 proteins have been sequenced (gene data bases = many more)
  • E. coli (human intestinal bacteria) makes about 3,000 proteins 
  • humans make about 100,000 different proteins w 25,000 genes ?      







4 levels of protein structure are recognized
 primary sequence  linear sequence of aa's from N-terminal to C-terminal


 secondary structure  regular, recurring orientations of aa's in a peptide chain
 due to H-bonds    =   alpha helix  & beta sheets
 tertiary  3D - conformational shape due to 
            weak electrostatic interactions with other atoms
      shape of most proteins  is GLOBULAR  [figure*]
 quarternary            2 or more different polypeptides or subunits
     interacting to give a unique 3D spatial relationship







   PRIMARY SEQUENCE  -  Some Consequences...                

 Polymorphism many proteins may vary in primary aa sequence, but
                      can have the same biological catalytic activity
 ex:    peroxidase/catalase enzymes       H2O2 --> 2 H2O + O2
  inter-specific : between species - different aa sequences
  intra-specific : within a species (liver vs. kidney)
 Invariants primary sequences don't vary significantly...  ubiquitin & histones 
some aa sequences determine intra-cellular location for a protein
              Signal Sequences,   prosthetic binding sites,     etc..
Proteins related by common evolutionary history...
  all evolved in a related fashion, but in different species...
  may perform similar cellular functions in the diff species.   
     ex: Hb & cyto-C     in ducks & chickens    =    2 variants
                                     in yeast & horses       =  48 variants









Secondary Level - a regular recurring pattern/shape in proteins due to H-bonds

 alpha Helix... is the peptide backbone wound around a long axis core*

forms a rigid cylinder - right handed helix - (counterclockwise) 
R-groups radiate outward
3.6 aa per 360o turn
helix is formed by H-bonds*
                       H of N (of one aa)   &  -C=O  (of 4th aa down)
of all aa's in globular proteins are in alpha  helicies*

 Beta sheet...

a linear extended ZIG-ZAG* pleated sheet
formed by H-bonds...  intra- & inter-chain            figure*







Tertiary Level 3D shape of a protein...         HIV's  Protease ribbon structure
Endonuclease often assumes the most stable conformation  (under given conditions)
 involves weak forces*    (figure                 
       H-bonds, hydrophilic & hydrophobic interactions
       & some stronger bonds as:   ionic bonds  &  disulfide bonds
figures* of  alpha  &  beta  sheets folded into tertiary level structures
3D shape of a protein....   collagen & Hb*   &   compare 30 & 40*
between more than one polypeptide or subunits of a protein
         ex:  hemoglobin...    hemoglobin*   &    ribbon*

       summary of all four levels    figure*  &   table*
    Concept Activity - 5.4 - Protein Structure*               structure's relatedness to functionality-ebola















 ENZYME...   (Gk)    "in   yeast
 discribed in 1878 Pasteur's lab <--> 1st called  "ferments"  (for fermentation)
 1st enzyme crystallized in 1926 by Sumner - was Urease
breaks down urea* into NH3 & CO2
 enzymes regulate metabolic reaction rates in cells, i.e., they control metabolism
          "are molecules (mostly protein) that accelerate or catalyze
            chemical reactions  [ A ---> B ]  in cells,
            by breaking old covalent bonds and forming new covalent bonds"
 they're biological catalysts... but, they differ from chemical (metal) catalysts :
     1. have complex, specific structures (a unique primary sequence of aa's = 3D shape)
     2. act only upon a specific substance (substrate)
     3. do not change the direction (
fig 8.14 energetics*) of a chemical reaction  [inertia*]  
     4. function by lowering Ea* and therefore speeding up a reaction   
                                                                             Concept Activity Chapter 8.4 - How Enzymes Work*





Some Properties of Enzymes:
    besides a protein part, many enzymes require cofactor or coenzyme...
protein to hold coenzyme in proper orientation to catalyze "bond breaking"


 small inorganic ions that helps catalyze reactions
              Cu+2 ;  Mg+2 ;  Mn+2 Fe+2  etc...


 small, organic, non-protein ligands   [ such as NAD+ a B-vitamin ],
 which catalyze rx most often by...
         gains/loses e- ;   transfer groups ;   break bonds.
      Reaction path     E + S <---> ES <---> E + P            catalytic cycle of an enzyme*
   ex:   hydrolysis of sucrose*    &    sucrase reaction*(fig 5.16)
      Active site  portion of enzyme protein that attaches to the substrate
         by means of weak chemical bonds
                       (H-bonds, ionic bonds, hydrophobic forces, etc...)
         some theories on the 3D-active site SHAPE
                                                                            Concept Activity Chapter 8.4 Enzyme lab                end 8






         √  enzymes are proteins with a specific 3-D shape,
             that binds substrate to its ACTIVE SITE
                           lock & key*    vs.     induced fit*
         √  shape of enzyme is critical to its ability to convert A ---> B
             change an enzyme's 3D shape  &  it won't bind substrate

   What will change shape of a protein [active site] & can thus lead to denaturation*?
            1.  temp - increases kinetic motion, breaks H-bonds
            2.  pH - changes ionic charges = alters shape
            3.  inhibitors - chemicals that bind to enzyme
                                  & change its activity  [competitive/non-competitive]
            4.  poisons - organo-phosphorous compounds
                                 (many insecticides) bind to enzymes of nervous system & thus kill





ENZYME KINETICS - a way of describing the physical properties of enzymes...
                 as mathematical and/or graphical expression of reaction rates of enzymes
     Catalase          2 H2O2 ----> 2 H2O + O2            A  -->  B + C
    manometric enzyme measurements* 

   Concept Activity
     - Chapter 1.3

An introduction to  Graphing*
Investigation Activity - Process in Science
     - Chapter 8.4

How is Rate of Enzyme Catalysis Measured*

         rates = ml of O2 per min      a) 0.40      b) 0.80      c) 1.20


Characteristic Graphical Curves of Enzyme Activity
     or how to determine if a cellular reaction  A > B  is enzymatic ???
     1.   Rate           Vs.    [ E ]*                       is a linear line
  (0.8 ml O2/min)    
     2.   Rate          Vs.     pH                          reveals an optimum
     3.   Rate          Vs.     Temperature         reveals an optimum
     4.   Rate          Vs.     [ S ]*                      shows a saturation curve
most definitive curve of enzyme activity 




MICHAELIS-MENTEN Enzyme Curve    [a Graphical Plot of enzyme activity]
a graphical plot of rate (amount of product per unit time*)  vs   [ S ] shows the curve saturates*
           ... reaches a point where [S] equals maximum velocity of rx = Vmax 
Km is the  substrate concentration  at which the rate is
                           one-half  the maximal velocity (Vmax)    
(in graph below*   Km = 2 mg)
               it's a rough measure of affinity of enzyme for its substrate
               it's the amount of [S] needed to reach 1/2 Vmax
Compare 2 enzymes*: G-6-phosphatase* [Km = 1 mg]  &  G-1-phosphatase* [Km = 25 mg]
Use of M
& M Curves - especially with environmental inhibitors   Enzyme Inhibition*
      competitive inhibition... binds to active site ...reversible   lower  Km       same  Vmax
        noncompetitive inhibition... binds to an allosteric site         same  Km      lower  Vmax






       plot of rate (amount of product per unit time) vs [S] at constant [enzyme]


figure of saturation*

1.  saturates... at high [S] = Vmax (maximum velocity) in graph        Vmax = 10mg per min   @ a constant [E]
2.  Km = Michaelis Constant is the substrate concentration at which rate is one-half the maximal velocity (Vmax)
                                             in graph above         Km = 2 mg of substrate
3.  Km is measure of affinity of enzyme for a substrate;    it is amount of [S] needed to reach 1/2 Vmax









Naming of Enzymes      mostly historical :  substrate + "ase"
                                      sucrase, , catalase, mallerase (?), but what about trypsin... etc
    International Union Biochemistry and Molecular Biology
          - 4 digit Nomenclature Committee Numbering System  [EC] 
               1st    Major Class of Activity     (only 6 classes recognized)
               2nd   Subclass (type of bond acted upon)
               3rd   Subclass (group acted upon, cofactor required, etc...)
               4th   Serial number ... sequence order





MAJOR CLASSES            [ Enzyme Commission Name Database]

1. Oxidoreductases
 catalyze oxidation reduction reactions, often w coe NAD+/FAD
     Alcohol dehydrogenase [EC
          ethanol + NAD+ -----> acetaldehyde + NADH
2. Transferases  catalyze the transfer of functional groups
     Glucokinase (hexokinase) [EC
          glucose + ATP -----> glucose-6-phosphate + ADP
3. Hydrolyases  catalyze hydrolysis - adds water across bonds as C-C
     Carboxypeptidase A   [EC] 
          [aa-aa]n + H2O -----> [aa-aa] n-1 + aa
4. Lyases  add or remove functional groups to C=C bonds
     Pyruvate decarboxylase [EC] 
          Pyruvate -----> acetaldehyde + CO2
5. Isomerases 
 catalyze isomerizations - change from one isomer to another
     Maleate isomerase [EC] 
          maleate -----> fumarate     (cis-trans isomerization)
6. Ligases           condensation of 2 substrates with splitting of ATP
     Pyruvate carboxylase [EC]
          pyruvate + CO2 + ATP -----> Oxaloacetic acid + ADP + P

         University of Miami Page  |  Biology's Page  |  Dr. Mallery's Page  |  On-Line Testing Center.
           copyright c2007, Charles Mallery, Department of Biology, University of Miami, Coral Gables, FL 33124  
           Biology 150: Last Update - 29 September, 2008





















      Enzyme Inhibition - is a reduction in enzyme activity
                  due to action of a non-substrate molecule on an enzyme's reactivity [shape = binding]...

*              inhibitor binds to active site, because it looks like substrate
                                      reversible;  can be removed by excess substrate
                                                         i.e., substrate can out compete inhibitor for active site
                                      raises the Km value,  but has same Vmax + an ex: Viagra*

                 Noncompetitive          inhibitor binds to an allosteric site, [not active site]
                                               ...isn't reversible; 
                                               ...thus inhibitor removes a fixed amount of enzyme - lowers Vmax
                                      has same Km, but lowers the Vmax

      Feedback inhibition
*  an ex:  an end product inhibits an initial pathway enzyme
                                                    by altering efficiency of enzyme action, probably by...

      Regulation of Metabolism  via  Enzyme Cooperativity & Allosteric Regulation   
               regulates enzyme activity by changing protein shape conformations
     allosteric sites and active vs. inactive forms*  &  regulation*