Enzymes

Enzyme as protein
Dr.Samina Haq

Quantitative and qualitative test for protein and amino acids
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Qualitative test

Ninhydrin test
Biuret test
Xanthoproteic test
Millons test
Hopkins-cole test
Nitroprusside test

Quantitative test
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Spectrophotometric assay Protein shows maximum absorbance at 280nm due to presence of tyrosine and tryptophane. Biuret test shows 540nm
Lowry test shows 750nm

Ninhydrin Test
• Amino acid containing a free amino group and a free carboxylic acid group that react together with ninhydrin to produce colured product. • Protein also contain free amino group on the alpha-carbon and can react with ninhydrin to produce blue purple product.
• When the amino group is secondary the condensation product is yellow.

Biuret Test
• It positively identifies the presence of protein in solution with a deep voilet colour.Biuret H2NCONHCONH2 reacts with copper(11)ions in a basic solution to form a deep voilet complex.

Xanthoproteic Test
• The amino acids that contain benzene ring like tyrosine and tryptophan undergo nitration in this test and gives yellow colour.

Millon’s Test
• It is specific for tyrosine, the only amino acid that contain a phenol group on which a hydroxyl group is attached. It gives red precipitate.

Hopkins_cole Test
• It is specific for tryptophan, the only amino acid containing an indole group.The indole ring reacts with glyoxylic acid in the presence of a strong acid to form a voilet cyclic product.

Nitroprusside Test
• It is specific for Cysteine, the only amino acid containing sulfahydryl group. Forms red complex.

Classification and Nomenclature of
Enzymes(IUBMB classification)
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Enzymes are commonly named by adding a suffix’’ase’’ to the root name of the substrate molecule it is acting upon.
Lipase catalysis hydrolysis of lipid triglyceroids. Sucrase= act on sucrose to form glucose and fructose.
The latest systamic nomenclature is known as
International Enzyme commission (IEC)system is based on type of reaction catalyzed.
Enzymes are classified at 4 levels which developed the
EzCatDB data base.
Basic reaction(R) reaction type (Class)
Ligand group involved in catalysis (L) (Sub Class)
Type of catalytic mechanism(C) (sub sub Class)
Residue/Cofactors located on Protein (P)

Enzyme Classified as Four digits separated by a point.

Enzyme Classification Biochemical Properties
Number
Oxidoreductase Act on Many chemical grouping, to add or
E.C1

remove hydrogen atoms or electrons
()xidation reduction, may require NAD,FAD

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Systemic name:
E.C 2

Transferase

Transfer functional groups to donor and acceptor molecules.Kinases are specialized transferase that transfer ATP to other molecules. DNA methyl transferase.

E.C 3

Hydrolases

Cleavage of bond between C and other group by addition of water.(Hydrolysing it)

E.C 4

Lyases

Non hydrolytic Cleavage of C-C,C-S,C-N bond.Add water.ammonia or carbon dioxide across the double bondor remove these elements to produce double bonds.

E.C 5

Isomerases

Convert one isomer to another by transfering one group to another.within the same molecule

E.C 6

Ligases

Catalyzes two reactions in which two chemical groups are joined or ligated with the use of energy.ATP.

SubClass Name

E.C 1

E.C1.1
E.C 1.2
E.C 1.3

Enzyme
File
type
Oxidoreductase
Sub sub cLass upto 50
Acting on the CH-OH group Up to of donors
50
Acting on the aldehyde or
Up to oxo group
50
Acting on CH-CH group of Up to donar 50