Enzymes

Proteases
Protease is the digestive enzyme needed to digest protein. The enzyme Proteases also called proteolytic enzyme & PeptideCutter. Protease refers to a group of enzymes whose catalytic function is to hydrolyze (breakdown) peptide bonds of proteins. They are also called proteolytic enzymes or proteinases. Proteases differ in their ability to hydrolyze various peptide bonds. Each type of protease has a specific kind of peptide bonds it breaks. Examples: Pepsin, Trypsin, Chymotrypsin, papain.

Mechanistic Sets of Proteases

set feature inhibitor examples function

Serine protease

active site serine

fluorophosphates

trypsin thrombin plasmin coccoonase subtilisin acrosin

digestion blood coagulation lysis of blood clots mechanical digestion sperm penetration

Cysteine protease

active site cysteine

iodoacetate

papain strept. proteinase cathepsin B

digestion digestion intracell. digestion

Acid protease

acidic pH optimum

diazoketones

pepsin chymosin

digestion milk coagulation

Metalloproteases

Zn2+, Zn2+, Ca2+

o-phenanthroline o-phenanthroline

carboxypeptidase thermolysin

digestion digestion

Proteases
What do proteases do?

Proteases cleave proteins by a hydrolysis reaction the addition of a molecule of water to a peptide bond

Proteases belong to the class of enzymes known as hydrolases, which catalyse the reaction of hydrolysis of various bonds(peptide bond, ester bond etc.) with the participation of a water molecule. Proteases are involved in digesting long protein chains into short fragments, splitting the peptide bonds that link amino acid residues.

Occurrence
Proteases occur naturally in all organisms. These enzymes are involved in a multitude of physiological reactions from simple digestion of food proteins to highly regulated cascades (e.g., the blood-clotting mechanism). Proteases can either break specific peptide bonds (limited proteolysis), depending on the amino acid sequence of a protein, or break down a complete peptide to amino acids (unlimited proteolysis). Bacteria also secrete proteases to hydrolyse (digest) the peptide bonds in proteins and therefore break the proteins down into their constituent monomers. Bacterial and fungal proteases are particularly important to the global carbon and nitrogen cycles in the recycling of proteins, and such activity tends to be regulated in by nutritional signals in these organisms.

How Protease Deficiency Can Affect Your Health

Acidity is created through the digestion of protein. Therefore a protease deficiency results in an alkaline excess in the blood. This alkaline environment can cause anxiety and insomnia. In addition, since protein is required to carry protein-bound calcium in the blood, a protease deficiency lays the foundation for arthritis, osteoporosis and other calcium-deficient diseases. Because protein is converted to glucose upon demand, inadequate protein digestion leads to hypoglycemia, resulting in moodiness, mood swings and irritability. Protease also has an ability to digest unwanted debris in the blood including certain bacteria and viruses. Therefore, protease deficient people are immune compromised, making them susceptible to bacterial, viral and yeast infections and a general decrease in immunity

Functions of proteases
Proteases are used throughout an organism for various metabolic processes. Acid proteases secreted into the stomach (such as pepsin) and serine proteases present in duodenum (trypsin and chymotrypsin) enable us to digest the protein in food. Proteases present in blood serum play important role in bloodclotting, as well as lysis of the clots, and the correct action of the immune system. Proteases determine the lifetime of other proteins playing important physiological role like hormones, antibodies, or other enzymesthis is one of the fastest "switching on" and "switching off" regulatory mechanisms in the physiology of an organism. Proteases are part of many laundry detergents.

Classification
Exopeptidases Some of them can detach the terminal amino acids from the protein chain is called exopeptidases. eg. Amino peptidases, carboxypeptidase. Endopeptidases Some of them can attack internal peptide bonds of a protein chain is called endopeptidases. eg. Trypsin, chymotripsin, pepsin, papain.

Classification
Proteases are divided into four major groups according to the character of their catalytic active site. Serine proteinases Eg: Trypsin,Chymotripsin. Cysteine proteinase eg: Bromelain (pineapple), Papain(papaya). Aspartic proteinases eg: Pepsin Metalloproteinases eg: Collagenase.

Serine proteases
Serine proteases (serine endopeptidases) are enzymes that cleave peptide bonds in proteins, in which serine serves as the nucleophilic amino acid at the active site. They are found in both eukaryotes and prokaryotes. eg: Chymotrypsin and Trypsin. In humans, they are responsible for co-ordinating various physiological functions, including digestion, immune response, blood coagulation and reproduction.

Cysteine proteases
Biological importance Cysteine proteases play an important role in every aspect of physiology and development in plants. In humans they are responsible for immune responses, prohormone processing, and extracellular matrix remodeling important to bone development. In several traditional medicines the fruits of papaya, pineapple and fig are widely used for treatment of intestinal worm infections in humans. Cysteine proteinases isolated from these plants have been found to have high proteolytic activities that are known to digest cuticles, with very low toxicity.

Aspartic proteases
Aspartic proteases are a family of protease enzymes that use an aspartate residue for catalysis of their peptide substrates. Aspartyl proteases play an important role in several aspects of our overall health and physiology, including blood pressure (renin), digestion (pepsin and chymosin). Aspartate proteases include the digestive enzyme pepsin Some proteases found in lysosomes the kidney enzyme renin HIV-protease.

Metalloproteinases
Metalloproteinases (metalloproteases) constitute a family of enzymes from the group of proteases, classified by the nature of the most prominent functional group in their active site. These are proteolytic enzymes whose catalytic mechanism involves a metal. Most metalloproteases are zinc-dependent, but some use cobalt. The digestive enzyme carboxypeptidase is a classic example of the zinc proteases. Zinc proteases (metalloproteases) include: digestive enzymes carboxypeptidases matrix metalloproteases (MMPs), secreted by cells one lysosomal protease.

Protease Applications in Food Processing

Basic rationale: Proteases are a powerful tool for modifying the properties of food proteins. Improved digestibility Improved solubility Modified functional properties: emulsification, fatbinding, water-binding, foaming properties, gel strength, whipping properties, etc. Improved flavor & palatability Improved processing: viscosity reduction, improved drying, etc.

Protease Applications in Food Processing

Proteases are also used in a wide range of foods & food processing applications. Dairy: milk coagulation, flavor development Baking: gluten development Fish & seafood processing: fishmeals, enhanced oil recovery, aquaculture Animal protein processing: improved digestibility, reduced allergenicity, improved flavor, meat tenderization Plant protein processing: improved functionality & processing, generation of bio-active peptides. Yeast hydrolysis: flavor compounds.

Nonfood Protease Applications

Medicine Pharmacology & drug manufacture Laundry & dishwashing detergents (#1) Hard surface cleaning formulations Contact lens cleaning formulations Waste treatment Industrial applications Fermentation (fuel EtOH, etc.) Chondroitin & heparin production Animal feed additives Digestive supplements

Carbonic anhydrases
The carbonic anhydrases (carbonate dehydratases) form a family of enzymes that catalyze the rapid interconversion of carbon dioxide and water to bicarbonate and protons. Carbonic anhydrases are enzymes that catalyze the hydration of carbon dioxide and the dehydration of bicarbonate: CO2 + H2O <-----> HCO3- + H+ One of the functions of the enzyme in animals is to interconvert carbon dioxide and bicarbonate to maintain acid-base balance in blood and other tissues, and to help transport carbon dioxide out of tissues. The carbonic anhydrases making fast reaction faster.

Structure and types of carbonic anhydrase

Several forms of carbonic anhydrase occur in nature. In the best-studied -carbonic anhydrase form present in animals, the zinc ion is coordinated by the imidazole rings of 3 histidine residues. The primary function of the enzyme in animals is to interconvert carbon dioxide and bicarbonate to maintain acid-base balance in blood and other tissues, and to help transport carbon dioxide out of tissues. Plants contain a different form called -carbonic anhydrase, which is a distinct enzyme, but participates in the same reaction and also uses a zinc ion in its active site. In plants, carbonic anhydrase helps raise the concentration of CO2 within the chloroplast in order to increase the carboxylation rate of the enzyme RuBisCO. This is the reaction that integrates CO2 into organic carbon sugars during photosynthesis, and can use only the CO2 form of carbon.

Reaction
The reaction catalyzed by carbonic anhydrase is: (in tissues - high CO2 concentration) . The reaction rate of carbonic anhydrase is one of the fastest of all enzymes, and its rate is typically limited by the diffusion rate of its substrates. The reverse reaction is also relatively slow, which is why a carbonated drink does not instantly degas when opening the container. However it will rapidly degas in the mouth when it comes in contact with carbonic anhydrase that is contained in saliva. An anhydrase is defined as an enzyme that catalyzes the removal of a water molecule from a compound, and so it is this "reverse" reaction that gives carbonic anhydrase its name, because it removes a water molecule from carbonic acid. (in lungs and nephrons of the kidney - low CO2 concentration, in plant cells)

Functions of carbonic anhydrase

While the main carbonic anhydrase function is working as a catalyst for the conversion of water and carbon dioxide (making it essential to the breathing process and the spread of oxygen to the cells in the body), it also plays other roles. These include: Plays a key role in the digestion process. The enzyme maintains the alkaline level of pancreatic juices while retaining the salivas neutral level to make sure that the body is digesting food as it should. Helps in the treatment of glaucoma. Carbonic anhydrase inhibitors are actually used to treat glaucoma. One of the possible causes of this eye condition is fluid build-up in the eye area, which can cause damage to the optic nerves.

 The enzyme can also be used to treat hypertension Works as a diuretic. By reducing the amount of salt and bicarbonate absorbed by the body, the enzyme also has diuretic effects that can help the body flush out harmful toxins in the system. Maintains pH and fluid homeostasis in the body. The enzyme is responsible for regulating the bodys pH level as well as fluid balance of the body. Carbonic anhydrase function is many and varied, depending on where the enzyme is located. Should there be abnormalities involving the enzyme, such as the body having too much or too little of it, the body can suffer, with possible problems including poor digestion and kidney failure.

Restriction enzymes
A restriction enzyme is an enzyme that cuts double-stranded or single stranded DNA at specific recognition nucleotide sequences known as restriction sites. Restriction enzymes are called molecular scissors. Nucleases are further described by addition of prefix endoor exoto the name. Endonucleases break nucleotides at the middle and exonucleases break the nucleotides at the end of the molecule.

Cut and ligate 2 DNAs with EcoRI ---> recombinant DNA

 Restriction enzymes recognize a specific sequence of nucleotides, and produce a double-stranded cut in the DNA. These cuts are of two types: STICKY ENDS

BLUNT ENDS.

Restriction enzymes
Recognizes specific base sequences in double-helical DNA and cleave, at specific places, both strands of a duplex containing the recognized sequences. Restriction enzymes recognize specific bases pair sequences in DNA called restriction sites and cleave the DNA by hydrolyzing the phosphodiester bond. Cut occurs between the 3 carbon of the first nucleotide and the phosphate of the next nucleotide. Restriction fragment ends have 5 phosphates & 3 hydroxyls.

Restriction enzyme

TYPES OF RESTRICTION ENZYMES Restriction endonucleases are categorized into three general groups. Type I Type II Type III These types are categorization based on: Their composition. Enzyme co-factor requirement. the nature of their target sequence. position of their DNA cleavage site relative to the target sequence.

 Type I restriction enzymes were the first to be identified and are characteristic of two different strains of E. coli. The recognition site is asymmetrical and is composed of two portions one containing 3-4 nucleotides, and another containing 4-5 nucleotides separated by a spacer of about 6-8 nucleotides. Type II restriction enzymes - They are composed of only one subunit. Their recognition sites are usually undivided and 4-8 nucleotides in length, they recognize and cleave DNA at the same site. Type II restriction enzymes - Type III restriction enzymes recognize two separate non-palindromic sequences that are inversely oriented. They cut DNA about 20-30 base pairs after the recognition site. These enzymes contain more than one subunit.

APPLICATION OF RESTRICTION ENZYMES They are used in gene cloning and protein expression experiments. Restriction enzymes are used in biotechnology to cut DNA into smaller strands in order to study fragment length differences among individuals (Restriction Fragment Length Polymorphism RFLP).

Nucleoside Phosphate Kinase

It is an enzyme that catalyzes the phosphoryl group exchange between nucleotides without promoting hydrolysis. It is also known as phosphotransferase. It is a type enzyme that transfers phosphate group from high energy donor molecule such as ATP to specific target molecule i.e., substrate. This process is termed as phosphorylation. All kinases require a divalent metal ions such as Mg2+ and Mn 2+.

Thus, the two substrates of this enzyme are ATP and nucleoside phosphate, whereas its two products are ADP and nucleoside diphosphate.