EXPERIMENT 4&5

Xanthoproteic Test

• Used to detect aromatic amino acids
  - Tyrosine
  - Tryptophan

• Reaction w/ Nitric Acid

• Result: Yellow

Hopkins-Cole Test

• Detect presence of tryptophan

• Result: Purple or Violet ring at the surface of the liquid (Positive)

Ninhydrin Test

• Used to detect amino acids and proteins by reacting w/ free amino groups
  (-NH2)

• Result: Deep purple-blue dye called Ruhemann's purple.

Biuret Test

• Used to detect peptide bonds in proteins

• Result:
  - Positive = Purple
  - Negative = Blue

Millon’s Test

• Used to detetct phenol compounds in protein

• Result: Red/Pink Colored Precipitate

Sakaguchi Test

• Used to detect presence of arginine (guanidinium group)

• Result: Red Colored Complex

Nitroprusside Test

• Used to detect presence of free sulfhydryl groups (-SH)
  - Cysteine

• Result: Red colored Complex

Enzyme

• Catalyst for biochemical reaction

Simple Enzymes

• Composed of exclusively proteins

• entire catalytic structure relies solely on complex amino

  acid chains

Conjugated Enzymes

• non-protein component + core protein structure

• Results biochemically active and capable catalyst

Apoenzyme

• The pure protein portion of a conjugated enzyme.

• Biochemically inactive w/o its required co-factor

Cofactor

• Prosthetic Group

• Tightly bound organic cofactor that permanently

  attaches to the enzyme.

  
  

Coenzyme

• Specialized cofactor

• small organic molecule that serves as a necessary cofactor

  in a conjugated enzyme system

Holoenzyme

• Apoenzyme + Cofactor

• Produces true catalytic activity

Zymogen

• Inactive precursor that posses initial catalytic activity

• Require biochemical change to reveal active site and become fully functional

• Critical regulatory safety mechanism in the body.

Major Enzyme Classification

• Oxidoreductases

• Transferases

• Hydrolases

• Lyases

• Isomerases

• Ligases

Oxidoreductase

• Function: Catalyzes critical oxidation-reduction reactions,

  transferring electrons between molecules.

• Example: Reductase, Oxidase, Dehydrogenase.

Transferases

• Function: Facilitates the intermolecular transfer of a

  specific functional group from one molecule to another.

• Example: Transaminase, Kinase.

Hydrolase

• Function: Catalyzes a hydrolysis reaction in which the

  addition of a water molecule to a bond causes that bond

  to break.

• Example: Proteases, Carbohydrases (like Salivary

  Amylase), Lipases.

Lyase

• Function: Catalyzes the addition of a group to a double

  bond, or conversely, the removal of a group to form a new

  double bond.

• Example: Dehydratase, Hydratase, Carboxykinase.

Isomerase

• Function: Facilitates the intramolecular transfer of a

  functional group, rearranging the molecule's structure

  without altering its chemical formula.

• Example: Isomerase, Racemase, Mutase.

Ligase

• Function: Catalyzes the direct bonding together of two

  distinct molecules into one single, larger molecule, often

  requiring ATP

• Example: Synthase

Active Site

• relatively small, highly specific part of the enzyme's

  overall protein structure

• Involved in catalysis

• precise region that binds substrates and essential cofactors.

Enzyme-substrate complex

• Critical intermediate reaction species

• temporarily formed only when a substrate successfully binds

  to the active site of an enzyme.

Lock and Key Model

• Rigid Geometry
  - only substrates with a perfectly complementary

  geometry can be accommodated at such a site (Specific Key)

Induced-Fit Model

• Dynamic Flexibility
  - Allows for small, dynamic conformational changes in the shape or

  geometry of the active site to perfectly accommodate and tightly

  bind a specific substrate upon contact.

Thermal Kinetics

• Increasing temperature generally increases the rate of enzymatic

  reaction by increasing the number of enzyme-substrate collisions.

• Optimum Temperature
  - enzyme exhibits maximum activity.
  -Excessive heat beyond this point causes rapid protein denaturation and total activity loss.

Optimal Acidity/Alkalinity

• Optimum pH
  - the specific pH at which an enzyme exhibits

  maximum catalytic activity.

• Deviations from this narrow pH window alter the ionization states of

  amino acid side chains within the active site, disrupting crucial salt

  bridges and leading to denaturation.

Competitive Inhibitor

• A molecule that closely resembles the

  substrate and directly competes with the substrate for access to

  the active site.

Non-Competitve Inhibitor

• Binds to a different area of the enzyme (allosteric site), triggering a shape change that prevents the binding of the substrate entirely.