Protein - a Species Specific polymer of amino acids with biological activity
An Overview of Proteins*view@home

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

Structure and Properties of all functional Proteins
PROTEINS - are  POLYMERs  of AMINO ACIDS with biological reactivity...                 N-gly-asp-leu-his-met-pro-phe-trp-tyr-lys-ser-val-C                 N--G---D----L---H---M---P----F---W--Y--K---U---V--C     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 can synthesize*    ??? all, half,  none.
STRUCTURE of Amino Acids                alpha amino acid*
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?
	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^ 20 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?

Polar Charged  ACIDIC      Polar Charged BASIC	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         figure*
Polar uncharged	includes SER, THR, CYS,  TYR, ASN, GLN                            are soluble in water, i.e., hydrophilic  (attract H-bonds)                 figure*    contain hydroxyl or amino functional groups
non-polar    (aliphatic)	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*

 
  PROTEIN STRUCTURE

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

Fred Sanger - 1958 Nobel prize for determining AA sequence of INSULIN*                         species variance in insulins*  &   synthetic analogs*  &   ADA

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   figure*

                                     Animation of the Structure of Proteins*^view@home

  
   PRIMARY SEQUENCE  - ex: 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. kidney)
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
Site  Specificity	some aa sequences determine intra-cellular location for a protein & activities:                     Signal Sequence*,   prosthetic binding sites*,     etc...
Homologous    Proteins	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

Tertiary Level	animation*  - is Native 3D shape [conformation] of a protein...
	a  protein folds into its most stable conformation  (in a given cell environment)
	involves weak electrostatic molecular forces*   or the     weak bonds*          including:   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 Urease 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"
cofactor	small inorganic ions that help catalyze electron [redox] reactions               Cu+2 ;  Mg+2 ;  Mn+2 ;  Fe+2  etc...
coenzyme	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 SHAPE*...
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  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?

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

figure*

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 [½] the maximal velocity (Vmax)                                (in the Michaelis Menten Enzyme curve* -->  Km = 0.08 mM)                it's the amount of [S] needed to reach ½Vmax                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  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            ...is reversible;  can be removed by excess substrate
                                                         i.e., substrate can out compete inhibitor for active site
                                               ...it 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
                                               ...it 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 1.2.3.4.]

1st    Major Class of Activity     (only 7 classes recognized*)                                    Enzyme Commission Numbers -  [EC  1.11.1.6]

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 ]

     <-- next presentation 

 




















































          SKIP ALL THE PRESENTATION MATERIAL BELOW

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
SDS-electrophoresis	Sodium dodecyl sulphate [SDS] - separates proteins via their mass (MW)      SDS binds to peptide bonds linear-izing a protein with (-) charges                  Fig1*   &     animation of SDSview@home
isoelectric focusing	proteins migrate thru pH gradient gels to their isoelectric point*,          i.e., pH where no net charge & then stop migrating
2-D electrophoresis	combines  isoelectric focusing  &  SDS-electrophoresis    procedures*    and        a sample compares leukemic cells*
Protein          Identification &          Quantification	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*Curve* ]

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                 copyright c2022    Last update - May 2022
                Charles Mallery,    Biology 150, Department of Biology,   U. of Miami,  Coral Gables, FL 33124