- a Species Specific polymer of amino acids with biological activity  

lysozyme - hydrolyzes
                                peptidoglycans in bacterial cell walls      


                         An Overview of Proteins*view@home












                   Proteins are more commonly Classified by  FUNCTION  'rather than by structure'

Enzymes  catalytic activity    A ------> B
Transport Proteins bind & transport ligand molecules (hemoglobins)
Storage Proteins ovalbumin (egg), ferretin (iron), casein (milk)
Defensive Proteins* provide protection:  antibodies (IgG),    fibrinogen,
    thrombin,  and  snake venoms
(digestive enzymes)
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
Hormonal-signal proteins coordination of physiological activities (insulin)
Receptor Proteins* respond to chemical stimuli: includes
receptors, transcription factors & enhancers
       nomenclature  early protein naming was based on solubility


    animation about protein functions*view@home










Structure and Properties of all functional Proteins
  PROTEINS - are  POLYMERs  of AMINO ACIDS with biological reactivity...
    made of alpha-L-amino acids (
commonly just 20 occur in cellular proteins)

               of the 20 of the common amino acids, how many amino acids
               do you think Humans
synthesize*    ??? all, half,  none.   
          STRUCTURE of Amino Acids
               alpha amino acid*
amino acid generic          
  an amino acid is a ZWITTERION... a molecule with 2 functional groups of opposite charges,

          aa's contain 2 functional groups: a carboxyl group (-COOH)  &   an amino group (-NH2)
          i.e.,  a weak base (NH2 = NH3+) and a weak acid (COOH = COO-).
                  & each of these functional groups are often ionized* at physiological pH
                  [weak acid:  a proton (H+) donor   &     weak base:  a proton (H+) acceptor].  

           Isoelectric Point - a pH where there is no net charge in a molecule. [NH3+] & [COO-]
           pK - the pH at which all the groups are 50% ionized & 50% non-ionized
            What is charge of glutamic acid at pH 3.0 ?        at pH 9.0 ?      (ans*)








  What is an alpha amino acid? 
amino acid generic NH2-group is bound to an asymmetric alpha carbon:
αlpha = 2HN-C-COOH  βeta = 2HN-C-C-COOH  -  γ-gamma = 2HN-C-C-C-COOH
β^                                                        γ^
different chemical  (R)  groups make the common aa's  &
                               these 20 ubiquitous & universal amino acids are found in all living systems.

  Remember amino acids are optical isomers: D or L  
                                Every amino acid (but one - glycine) exists in two optical isomeric forms,
                                         each with different arrangements of atoms in space.   
                                         i.e., 2 optical isomers are mirror images of each other  
                             but only the α-L-optical isomer aa's occur in biological proteins
... an anomaly of molecular evolution?









the properties of the various proteins depends on the chemical properties of the amino acids
     amino acid's are
            attached to
α-carbon, which defines its chemical & physical properties.
Polar Charged

Polar Charged
negatively charged amino acids - ASP & GLU
   R group contains a 2nd COOH that ionizes above pH 7.0   
positively charged amino acids - LYS, ARG, HIS
   R group with a 2nd NH2 that protonates below pH 7.0 
Polar uncharged includes SER, THR, CYS,  TYR, ASN, GLN                        
   are soluble in water, i.e., hydrophilic
  (attract H-bonds)
   contain hydroxyl or amino functional groups    
includes GLY, ALA, VAL, LEU, ILE, MET,  PHE, TRP, PRO,    figure*
   all contain only hydrocarbons R groups   =  
hydrophobic like lipids
AROMATIC (hydrophobic non-polars)    PHE & TRP     (TYR)    &    MET,  CYS
   all contain R groups with
    ring structures*         or         Sulfur*   
                                      Table of the Amino Acids*







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

peptide bond

forms a dipeptide*   or eventually a polypeptide*       
formed by a condensation reaction (removes water = dehydration synthesis)
         peptide bond is intermediate in length & strength between C-C  &  C=C
         thus a peptide bond is shorter & stronger than C-C and
         longer, but weaker, than  C=C, however it acts like one, thus
         there is  no free rotation (attached group in same plane)   figure*
         and the peptide bond is Flat and Planar* (with R-groups trans to each other)
                   non-protein examples of peptide bonds:
         Capsaicin (chili peppers active ingredient)
                               NutraSweet (dipeptide: L-aspartyl-phenylalanyl methyl ester) 

                             Review animation of Functional Groups in a tripeptide*view@home                         




















  • Variety of a linear polymer of amino acid sequences is infinite...  
  • A protein of 100 amino acids made with the 20 different known amino acids
          can have 20n (n = protein length = ~) different linear sequences for a protein
  • Average protein has 300 to 400 aa's  &  has a MW of 30,000 to 45,000d        
  • many often have a globular (spherical*) 3-D shape  &  are  negatively charged
  • some ≈ 1,000 proteins have been chemically sequenced (now done by gene data bases)
  • E. coli (a human intestinal bacteria) makes about 4,250 proteins 
  • Human Proteome Project has mapped with an estimated 30,057 human proteins
                                             made from only 17K protein coding genes
    (~3% of genome).      











4 levels of protein structure are recognized
          primary sequence  linear sequence of aa's from N-terminal to C-terminal
           NCC-NCC-NCC-NCC-NCC-NCC-NCC-NCC-NCC       figure*
          secondary structure  regular, recurring orientations of aa's within a peptide chain
         due to H-bonds forming  alpha helix  &  beta sheets    figure
          tertiary structure  3D - conformational shape due to 
         weak electrostatic interactions with other molecules

         make the shape of many soluble enzymes globular     figure*
   quaternary structure  2 or more polypeptides or protein subunits interacting to give
        to give a unique 3D spatial shape to a protein  

                                     Animation of the Structure of Proteins*view@home









primary sequence amination*view@home   &   Transthyretin*

Some properties of proteins due to Primary Sequences...
 Polymorphism some proteins may vary in their primary amino acid sequence, 
                                                  but can have the same biological catalytic activity

 ex:  catalase
/peroxidase  enzymes       2 H2O2 --> 2 H2O + O2
            inter-specific : between species - different aa sequences (cow vs. human)
            intra-specific : within a species (liver vs.
 Invariants primary sequences that do not vary significantly.   ex:   ubiquitin* & histones*
            ubiquitin (universal to eukaryotic cells) signals a proteins degradation
            histones - alkaline protein that bind with DNA
some aa sequences determine intra-cellular location for a protein & activities:
                    Signal Sequence
*,   prosthetic binding sites*,     etc...
Proteins related by common evolutionary history...
  i.e., proteins which are derived from common ancestor,
            can occur in different species, or same species (if 1 gene duplicated)...
            may perform similar cellular functions in the different species. 
                ex: Hb* & cytochrome-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...  animation* :the peptide backbone is wound around a long axis core*

forms a rigid cylinder* - a right handed helix - (counterclockwise) 
R-groups of amino acids radiate outward*
3.6 aa per 360o turn  - 1.5 Ao/residue    (5.4 Ao per turn)
                       1 angstrom =  1  x  10-10 meters     (or  0.1 nm)
diameter of helix is 1.2nm - same diameter as DNA major groove,
                      thus a-helices are common in DNA binding proteins.
alpha helix is formed naturally by H-bonds*
                       H of N (of one aa)   &  -C=O  (of 4th aa down)
helix is typically 10-15 residues long,
of a typical protein is in alpha helices   and   is beta sheet

  Beta sheet...


a linear extended ZIG-ZAG* pleated sheet conformation
formed by H-bonds...  intra- & inter-chain  figure* beta sheets*
beta sheets are usually 3 to 10 residues long







      Tertiary Level  animation- is Native 3D shape [conformation] of a protein...         
Endonuclease a  protein folds into its most stable conformation  (in a given cell environment)
involves weak electrostatic molecular forces*   or the     weak bonds*   
H-bonds, hydrophilic & hydrophobic interactions,
      & some stronger bonds as:   ionic bonds  & 
disulfide bonds   (animation of folding)
      some ex: tertiary structure of Human Glut-3 - a human glucose transport protein  
                    HIV's  Protease ribbon structure - an AIDS protein needed for virus
                  3D models of proteins structure using Lysozyme - antibacterial enzyme
   Quaternary Level animation* is the 3D arrangement of multiple protein subunits
  3D shape of a protein....   collagen & Hb*   &   comparing 30 & 40*
between more than one polypeptide or subunits of a protein

hemoglobin...  tertiary ribbon-view                            
Summary of all four levels: figure - table* & actual scaled size relations*
                                    animated  description of protein structure*view@home      
                                                animated review of protein structure & function* view@home                                                                                                                                                                                                       















 ENZYMEs... are proteins responsible for catalysis*     (term is from Gk - "in   leaven")   
 1st described in 1880's by Louis Pasteur  and  called  "ferments"  (their ability to ferment of sugars)
 1st enzyme crystallized in 1926 by Sumner - was 
breaks down urea* into NH3 & CO2
What is it that enzymes actually do?
   enzymes regulate metabolic reaction rates in cells, i.e., they control metabolism
       "are molecules
that accelerate catalytic chemical reactions  [ A ---> B ]  in cells,

        by breaking covalent bonds and/or forming new covalent bonds".
(most cellular catalysis is by proteins - ? but why not all?)
 they're biological CATALYSTS*... but, differ from classical chemical (metal-Fe) catalysts in that:
     1. have complex specific structures (a unique primary sequence of aa's = unique 3D shape)
     2. act only upon a specific substance (a substrate)
     3. do not change the direction
fig 8.14 energetics*) of a chemical reaction
     4. enzymes work by lowering Ea* bringing reactants together & thus speeding up* reaction rates*   








Some Terminology & Properties of Enzymology:
    besides a protein part, many enzymes may require a cofactor or coenzyme*... 
i.e., protein holds a coenzyme in proper orientation to catalyze "bond breaking - forming"


 small inorganic ions that help catalyze electron [redox] reactions
              Cu+2 ;  Mg+2 ;  Mn+2 Fe+2  etc...


 organic, non-protein molecules, which catalyze [redox] reactions
 many are
vitamins  such as NAD+*  (or vitamin-B3),
  that...  gains/loses e- ;   transfer groups ;   break bonds, etc...
      reaction path     E + S <---> ES <---> E + P      catalytic cycle of an enzyme
                       ex:   sucrase reaction*(fig 5.16)
      active site  portion of enzyme protein that attaches to the substrate
         by means of
weak chemical bonds
* to lower Ea...     
               (via H-bonds, ionic bonds, hydrophobic forces, etc...)
         some theories on the 3D-active site
                                                                                                                                                                end 8















Structure of ENZYME-SUBSTRATE Complex:
         √  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*?
       Cells regulate pH and tmperature via homeostasis, thus changes in...    
             1.  temp - increases kinetic motion, breaks H-bonds
             2.  pH - changes ionic charges, can alter shape
             3.  inhibitors - chemicals that bind to enzyme
                                & change its activity  [competitive/non-competitive inhibition]
             4.  poisons - organo-phosphorous compounds (many insecticides)
                                  bind to enzymes of nervous system & thus kill

   Example of a Mechanism of Action of an enzymr's Active Site - Serine Proteases*















ENZYME KINETICS - a way of describing the physical properties of enzymes...
           by mathematical and/or graphical expression of reaction rates of enzymes
     Catalase          2 H2O2 ----> 2 H2O + O2            A  -->  B + C
          to measure rate we could monitor the disappearance
 of A or appearance of product (B of C)
manometric enzyme measurements* 



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

but, how do we know if this reaction is catalyzed by an enzyme vs. an Fe catalyst?       

peroxide bubbles



Characteristic Graphical Curves of Enzyme Activity
     or how to determine if a cellular reaction  A > B  is enzymatic ???
          by analyzing the kinetics of the reaction.
     1.   Rate           Vs.    [ E ]*                       is a linear line, (same as metal catalyst)
  (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 describing 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 
the Enzyme Curve defines a new constant relative to many enzymes:           
Km is the  substrate concentration  at which the rate is
               one-half [] the maximal velocity (Vmax)  
(in the Michaelis Menten Enzyme curve* -->  Km = 0.08 mM)
               it's the amount of [S] needed to reach Vmax

               it's a "rough" measure of affinity of enzyme for its substrate
          Compare 2 enzymes*:    G-6-phosphatase [Km = 1 mg]  &  G-1-phosphatase [Km = 26 mg]
                                                                it takes much less substrate for G-6 to reach same kinetic point
                                                                thus G-6-phosphatase is more efficient than G-1-phosphatase.

Use of M
& M Curves - especially with environmental inhibitors   Enzyme Inhibition*
      competitive inhibition... binds to active site ...reversible  'raises Km       same  Vmax
        noncompetitive inhibition... binds to an allosteric site       same    Km      lower 













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

M&M curve



figure of enzyme saturation*

1.  SATURATES... at high [S]  = Vmax (maximum velocity) in graph 
                                                    Vmax = 62 uM per min   @ a constant [E]

2.  Km = Michaelis Constant is substrate concentration
                              when the rate is one-half maximal velocity (Vmax)
                                         in graph above       Km = 0.08 mM of substrate

3.  Km can indicate a measure of affinity of an enzyme for a substrate;
it is amount of [S] needed to reach Vmax  

























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

              Irreversible*         inhibitor covalently binds to enzyme permanently inactivating it

              Competitive*        inhibitor binds to active site, because it looks like substrate
                   animation   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]
                   animation            ...isn't reversible; 
                                               ...thus inhibitor removes a fixed amount of enzyme - lowers Vmax
                                      has same Km, but lowers the Vmax


      Regulation of Metabolism  via  Enzyme Cooperativity & Allosteric Regulation
             regulates enzyme activity by changing protein shape conformations


      ex:  Feedback inhibition*   an end product inhibits an initial pathway enzyme
          animation       by altering efficiency of enzyme action.
              many enzymes function by allosteric sites and regulation by active vs. inactive forms*
                     summation of Michaelis-Menten enzyme Regulations*   
















Two additional aspects of enzymes:     their nomenclature   &   how to isolate an enzyme.

    Naming of Enzymes      mostly historical :  substrate + "ase"
                                               kinase, sucrase,  catalase,  mallerase (?),
                                               but what about common names as trypsin, pepsin, ... etc
    International Union Biochemistry and Molecular Biology     -    Enzyme Commission

          - Nomenclature Committee established a 4 digit  Numbering System  [EC]
               1st    Major Class of Activity     (only 7 classes recognized*)
                                   Enzyme Commission Numbers -  [EC]

               2nd   Subclass (type of bond acted upon)
               3rd   Subclass (group acted upon, cofactor required, etc...)
               4th   Serial number ... sequence order
            <-- next presentation 









                            MAJOR ENZYME CLASSES...           
[ Enzyme Nomenclature Database ]

1. Oxidoreductases
 catalyze oxidation reduction reactions, often w coe NAD+/FAD
     Alcohol dehydrogenase
          ethanol + NAD+ -----> acetaldehyde + NADH
2. Transferases  catalyze the transfer of functional groups
     Glucokinase (hexokinase)
          glucose + ATP -----> glucose-6-phosphate + ADP
3. Hydrolases
 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
          Pyruvate -----> acetaldehyde + CO2
5. Isomerases 
 catalyze isomerizations - change from one isomer to another
     Maleate isomerase                              
          maleate -----> fumarate     (cis-trans isomerization)
6. Ligases           condensation of 2 substrates (often with splitting of ATP)
     Pyruvate carboxylase
          pyruvate + CO2 + ATP -----> Oxaloacetic acid + ADP + P
7. Translocases
 catalyze the movement of molecules across membranes  (ATP)
Phosphate transporter                          [EC]

*Common names of enzymes and their cellular roles*

     <-- next presentation 


















Protein Methodologies in Biological Chemistry
        - know the Basics,   master the Method,  be Persistent...  & repeat, repeat, repeat

FRACTIONATION - Methods for Isolation & Purification of "new" protein...
  Cell Homogenates 
 "grind & find"           homogenization of cells -->    methodology*         
  mortar/pestle,   blenders &  homogenizers                        
 subcell fractionation* - separation by size & density via centrifugations
             ultra-centrifuge --> 
speeds to 250,000xg  -->   collected fractions
                     Beckman Optima - cost $30K to $65,000+                     
 Column Chromatography:   separates proteins from other proteinss via flow thru media in a glass column

gel filtration

 exclusion chromatography...    concept figure 1*    
                Sephadex - porous carbohydrate gel* beads

                retards small MW proteins & passes large MW proteins
based on size.  

                  ion exchange

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


 polymeric beads with special ligands* (molecule of functional group), 
                which bind to specific proteins, but not other proteins








Electrophoresis - passage of charged protein mixture through a porous medium (gel) via its
                            electrical charge that results in
  separation of proteins by
size & charge*
   PAGE  polyacrylamide gel electrophoresis = PAGE*  in gel chambers & gels
protein fingerprints* can indicate evolutionary relations


 Sodium dodecyl sulphate [SDS] - separates proteins via their mass (MW)
     SDS binds to peptide bonds linear-izing a protein with (-) charges

*   &     animation of SDSview@home

isoelectric focusing

 proteins migrate thru pH gradient 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 compares leukemic cells*  
Identification &
  colorimetric reaction [Biuret & Bradford] where amount of
  color produced is proportional to amount protein present...
  "3 most cited science papers are on methods to measure proteins"
                   [ create  a Standard
* ]    

       back   next lec        
                 copyright c2022    Last update - May 2022

                Charles Mallery,    Biology 150, Department of Biology,   U. of Miami,  Coral Gables, FL 33124