Classification of enzymes

In 1961 the commission of Enzymes of the International Union of Biochemistry divided all enzymes into six main classes, each one of which is further subdivided into subclasses and sub-subclasses. The main classes are the following – I. Oxireductases, II. Transferases, III. Hydrolases, IV. Lyases, V. Isomerases, VI. Lygases (or synthetases). The numbers of classes, subclass, sub-subclass and one’s number of enzyme are the code of each enzyme. In many cases the old terminology is used: pepsin, trypsin, chymotrypsin, papain. We begin to study a different classes from more simple – third class – hydrolyses.

III. Hydrolases

These enzymes catalyze hydrolysis – addition of water molecule to the substrate and simultaneously decompose it, they catalyze all reactions with participation water. They have many subclasses (9).

1. protein hydrolyzing enzymes (proteinases or proteases or proteolitic enzymes. This subclass is divided into the 2 sub-subclasses: endopeptidases and exopeptidases. Endopeptidases catalyze the peptide bonds within the protein (from word “endo” which means inside) – proteins  (under influence of endopeptidases) n (polypeptide). The endopeptidases include a few types of enzymes: pepsin, trypsin, chymotrypsins, catepsins. Different endopeptidases affect hydrolysis at particular aminoacid residues. Pepsin is produced by the chief cells of the gastric mucosa as pepsinogen. Under influence of hydrochloric acid it turns into pepsin: pepsinogen (proenzyme)  pepsin (enzyme) + polypeptide. Pepsin is acid proteinase, so its active center are ions of carboxyl groups. Pepsin is very active enzyme: 1g of pepsin hydrolyzes 50 kg of ovalbumine during 2 hours. Besides pepsin splits caseinogen of milk:

caseinogen – 100 % (under influence of pepsin in adult; of rennin in young; of chymozyn in animal)  casein – 90 % + polypeptide (serum albumose) – 10 %. 1 g of pepsin turn into the curds 10000 liters of milk. Pepsin acts on the amino side of tyrosine and phenylalanine and the carboxyl side of dicarboxylic amino acids.

Trypsin is produced by the pancreatic cells as trypsinogen which in the intestine turns into trypsin under influence of enteropeptidase (enterokinase): trypsinogen  trypsin + hexopeptide. Trypsin is simple protein, its active center includes serine and hystidine; it acts on the carboxyl side of lysine and arginine. Chymotrypsin is produced by pancreatic cells as chymotrypsinogen which turns into chymotrypsin under the action of trypsin: chymotrypsinogen  chymotrypsin + polypeptides (from 2-10 residues). Its active center includes 2 2 amino acid residues – serine and hystidine. Chymotrypsin acts on the carboxyl side of tyrosine and phenylalanine. The ability of pepsin, trypsin and chymotrypsin act in different part of protein molecules to accelerate the hydrolysis of proteins. Catepsins are cellular endopeptidase. There are 6 types of these enzymes: A, B, C, D, E, Q. These are proteinase and have the enzymatic activity at pH 3-5 only. They are in lyposomes, and become active at the acidosis (inflammation, death, hypoxia) and hydrolyze a different proteins. This can to autolysis of cells and tissues. Exopeptidases catalyze the hydrolysis of a terminal peptide bonds. These are further subdivided into the following types: 1) polypeptidases – there are of two types aminopeptidases and carboxypeptidases: aminopeptidases are simple proteins (alfa-2 globulins). Ions of Zn, Mg, Mn activate these enzyme. These are in intestinal juice, cells and attack the protein molecule from the side containing a free aminogroup, yielding an amino acid and peptide smaller in size by one amino acid residue. The process may be repeated again and again.

Carboxypeptidase are conjugated proteins, coenzyme is Zn. These are in intestinal juice, pancreatic cells (procarboxypeptidase, trypsin is activator for this enzyme), different cells. Carboxypolypeptidase acts in the same way as aminopolypeptidase but attacks a polypeptide molecule from the side containing a free carboxyl group. Tripeptidases act on tripeptides. Dipeptidases act on dipeptide. These enzymes contain Zn and are in the cells, intestinal juice.

2. next subclass of hydrolyses is carbohydrases. These catalyze the hydrolysis of the glycosidic bonds: -C-O-C-. all these enzymes are simple proteins (see table).

Table 5. Glycosidases

Enzyme	Reaction	Localization
Alfa-amylase	Starch, glycogenmaltose	Cells, saliva, pancreatic juice
Gamma-amylase	Glycogenglucose	Liver
Maltase	Maltose2 glucose	Cells, intestinal juice
Lactase	Lactoseglucose + galactose	Cells of mamma, intestinal juice
Sucrase	Sucroseglucose + fructose	Intestinal juice
Cellulase	Cellulose	Intestinal juice of cow, horse

So, carbohydrases aren’t in gastric juice.

3. Esterases is next subclass and it is subdivided into the 2 sub-subclasses – lipases and phosphoesterases. 1. lipases act on triglycerides or neutral fats to liberate glycerol, fatty acids and mono- and diglycerides. 1) Lipase of gastric juice is active at pH 3-5 only. The pH of gastric juice of adult people is 1.5-2.0, therefore this enzyme is active in gastric of baby (child arms) when pH of gastric juice is 3-5. This enzyme acts on emulgated fat of milk only. 2) lipase of pancreatic juice is secreted by pancreatic cells in inactive form, one is activated by conjugated salts of bile acids. After activation lipase of pancreatic juice is very important enzyme in the digestion of fat. It acts on the triglycerides and splits them to fatty acids and monoglycerides.

3) intestinal lipase is secreted by mucous cells of intestine in active form and acts on the monoglycerides:

So, under the action of lipases in gastrointestinal tract triglycerides split to glycerol, fatty acids and monoglycerides. 4) cholesterase hydrolyzes cholesterol esters. 5) phospholipases A1, A2 hydrolyzes the ester bond at 1-st and 2-nd position in phospholipids. 2. Phosphoesterases hydrolyze the phosphoester bond. 1) phospholipases C and D hydrolyze the splitting the bond between glycerol and phosphoric acid (phospholipase C) and between phosphoric acid and nitro-containing matters (phospholipase D). 2) phosphatases – ATP-ase and glucoso-6-phosphatase.

Other hydrolases:

1. deaminases or aminohydrolases. These include adenase and guanase, which catalyze the following reactions: adenine + H2O  hypoxanthine + NH3; guanine + H2O  xanthine + NH3; hypoxanthine  xanthine  ureatic acid.

2. Deamidases or amidohydrolases catalyze the hydrolysis of amides and include urease, arginase, glutaminase and asparaginase, which catalyze the following reactions respectively: urea + H2O  CO2 + 2 NH3; arginine + H2O  ornithine + urea; Gln + H2O  Glu + NH3; Asn + H2O  Asp + NH3

3. phosphatases - a greit variety of phosphotases is found. These occur in many tissues: acid and alkaline phosphotases, phosphodiesterase, phosphorylase, pyrophosphatase.

4. Nucleases (polynucleotidases). These are present in the intestinal juice and tissues. These decompose nucleic acids (DNA and RNA) to mononucleotides. DNA and RNA  n (mononucleotides).

5. Nucleotidases – these enzymes occur in the intestinal juice and tissues and hydroluze mononucleotides and H3PO4.

6. Nucleosidases these catalyze the following types of reactions: nucleoside + H3PO4  free nitrogenous bases ( purine or pyrimidine) + sugar phosphate.

The use of hydrolases

1. enzyme substitution in digestive disturbances. The enzyme preparation used for the digestive disturbances contain proteinases, lipases and amylases of animal, vegetable and microbial origin. Pepsin and pancreatin (containing all enzymes of pancreatic juice) alone or along with dry bill powder are the most frequently used enzyme preparation in digestive disorders: for example, kreon, festal ect.

2. Blood coagulation. Trombin in used locally to stop bleeding urokinase and streptokinase are used to produce lysis of blood clots (tromboflebits, infaretus).

3. For lysis different hematomas, plastic surgery, trauma of face, box.

4. To increase blood supply to tissues. The enzyme kallicrein increases the formation of kinins from their precursors – kininogens, kinins produce vasodilatation and for this reason kallicrein is used in cases of impairment of the blood supply of tissues of the extremity and heart.

5. Locally applied enzyme preparations – 1) hyaluronidase – this enzyme hydrolyzes mucopolysaccharides, such as hyaluronic acid and chondroitin sulfatic and thus causes a loosing of the deep layers of the skin. It has been used for increasing the rate of fluid absorption from subcutaneous tissues and in the treatment of sprains, hematomas and thrombophlebitis.

6. Trypsin and chymotrypsin. These are used to degrade necrotic tissue, masses of pus, secretions and effusions thus producing a cleaning of the wounds and accelerating wound healing. Chymotrypsin is also used orally and by injection in these conditions (pneumonia, abscess, pleurit, pyelonephrite).

7. RNA-ase and DNA-ase. These are used in the form of streptodornase for the disintegration of pus.

8. Enzymes for cancer treatment. L-asparaginase has proved very useful in the therapy of limphoblastic leukemia especially in children. L-asparagine is substrate of this enzyme, is essential for the proliferation of cancer cells. Administration of L-asparaginase hydrolyzes asparagine and results in a decreased availability of asparagine to cancer cells and therefore their abnormally fast growth is arrested resulting in a remission.

9. Treatment of old age.

10. For treatment of renal deficiency is used urease

The use of hydrolases in industry

1. catepsins for treatment of meat.

2. In cheese dairy making.

3. In washing powder (proteases, lipases).

The general characteristic of transferases

Transferases catalyze the reactions of transfer of atoms groups from one substance to another. They’re mainly two-component enzymes, i.e. consist of apoenzyme and coenzyme. They’re contained in cells only.

On the depend on transported atoms groups they’re divided into:

1. aminotransferases (apoenzyme + phosphopyridoxal)

2. methyltransferases (apoenzyme + tetrahydrofolic acid)

3. acyltransferases (apoenzyme + HSCoA)

4. phosphotransferases, or kinases (no coenzyme)

Aminotransferases consist of apoenzyme and coenzyme (phosphopyridoxal). Phosphopyridoxal is vitamin B6 + H3PO4.

Aminotransferases catalyze the transamination:

alfa-ketoglutaric acid + alanine↔pyruvic acid + glutamic acid (enzyme – alaninetransferase. It is mostly located in liver, heart and skeletal muscles).

Another reaction of transamination is:

alfa-ketoglutaric acid + aspartic acid↔oxaloacetic acid + glutamic acid (enzyme – aspartateaminotransferase. It is mostly located in heart, liver and skeletal muscles).

The importance of transamination:

1. this is the pathway of formation of unessential amino acids

2. transamination associates protein metabolism with carbohydrate metabolism

3. this is initiate stage of amino acid catabolism

4. aminotransferases are specific enzymes which occur in some organs. Lesion of rgans results in exit of aminotransferases in blood. It is important for diagnostic aims.

The structure, role and representatives of methyltransferases

Methyltransferases consist of apoenzyme and coenzyme as tetrahydrofolic acid (THFA). THFA is reduced vitamin Bc which consists of paraaminobenzoic acid, glutamic acid and pteridine.

Methyltransferases catalyze the methylation. Vitamin B12 helps this reaction. The source of CH3-group is methionine:

Norepinephrine+ methionineepinephrine+homocysteine

ethanolamine+3methioninecholine+3 homocysteine

uracil+methioninethymine+homocysteine

The structure, role and representatives of acyltransferases

Acyltransferases consist of apoenzyme and coenzyme (HSCoA):

HSCoA consists of pantothenic acid, adenosine-3phosphate, thioethylamine, phosphate

Acyltransferases catalyze the acylation (transfer of residues of carboxylic acids):

Free fatty acid+HSCoA+ATPAMP+H4P2O7+acive fatty acid

The structure, role and representatives of phosphotransferases

Phosphotransferases, or kinases are one-component enzymes, but they have quaternary structure, i.e. consist of some subunits and therefore have got allosteric center. They catalyze phosphorylation. The source of phosphoric acid is ATP mainly. There are 2 types of phosphorylation – reversible and irreversible.

When the phosphoric acid joins with macroergic bond the phosphorylation is reversible.

Creatine+ATP↔creatinephosphate+ADP (enzyme is creatinekinase which is located mainly in skeletal muscles and heart). For example,

2ADP↔ATP+AMP (enzyme is adenilylkinase which is mostly located in skeletal muscles).

When the phosphoric acid joins without macroergic bond the phosphorylation is irreversible, e.g.:

Protein+ATPphosphoprotein+ADP (enzyme is proteinkinase which is located in all tissues)

Glucose+ATPglucose-6-phosphate+ADP

In some cases the source of phosphoric acid may be high energetic phosphorus containing substances. Such phosphorylation is named as substrate’s one:

Pyruvic acid+ATPphosphoenolpyruvate+ADP (enzyme is phosphopyruvatekinase, or pyruvatekinase)

3-phosphoglyceric acid+ATP↔1,3-diphosphoglyceric acid+ADP (phosphoglyceratekinase)

The general characteristic of lyases

Lyases catalyze the reactions of cleavage of substrates without water or addition of some atoms on the position of double bonds. They are two-component enzymes and occur in cells. They’re divided into:

1. decarboxylases of ketoacids (apoenzyme + thyamin diphosphate)

2. decarboxylases of amino acids (apoenzyme + phosphopyridoxal)

3. carbonic anhydrase (apoenzyme + zinc)

The structure and catalytic role of decarboxylases of ketoacids

Decarboxylases of ketoacids consist of apoenzyme and coenzyme as thyamin diphosphate. Thyamin diphosphate is vitamin B1 + 2H3PO4

Decarboxylases of ketoacids enter the multi-enzymic complexes, e.g. pyruvate dehydrogenase’s complex which catalyzes the reaction of oxidative decarboxylation of pyruvic acid. The 3 main enzymes enter this complex: pyruvatedehydrogenase, pyruvatedecarboxylase (PDC) and acetyltransferase. First enzyme catalyzes the dehydrogenation (removal of hydrogen) of pyruvic acid; PDC catalyzes decarboxylation (removal of CO2); acetyltransferase enters the CoA in product of reaction. This reaction results in formation of acethylCoA (active acetic acid).

The structure and catalytic role of decarboxylases of amino acids

Decarboxylases of amino acids consist of apoenzyme and coenzyme as phosphopyridoxal (vitamin B6 + H3PO4). Decarboxylases of amino acids catalyze the decarboxylation of amino acids that results in formation of biogenic, or proteinogenic amines:

5-hydroxytryptophanserotonin+CO2

Hystidinehystamine+CO2

Glutamic acidgamma-aminobutyric acid

Aspartic acidbeta-alanine+CO2

Phenylalaninephenylethylamine+CO2

Tyrosinetyramine+CO2

The structure and catalytic role of carbonic anhydrase

Carbonic anhydrase consists of apoenzyme and coenzyme as zinc. This enzyme catalyzes the reaction of cleavage of H2CO3 to CO2 and H2O: H2CO3↔H2O + CO2. The direction of this reaction depends on CO2 concentration, therefore this enzyme regulates the function of respiratory center

The general characteristic and classification of oxireductases

Oxireductases catalyze oxidative-reductive reactions. They’re two-component enzymes. Oxireductases may divide into 4 groups:

1. dehydrogenases

2. cytochromes

3. catalase and peroxidases

4. oxygenases

Dehydrogenases oxidize substrates by dint of their dehydrogenation (removal of hydrogen). They participate in biologic oxidation and reduction. There are pyridine and flavin (FP) dehydrogenases and ubiquinone. They may be primary and secondary. Primary DH-ases oxidize substrates. Secondary DH-ases oxidize reduced forms of other DH-ases. Cytochromes are heme-containing enzymes. They take part in transport of electrons. Catalase and peroxidase are heme-containing enzymes. They don’t participate in oxidative-reductive reations, but they cleave H2O2 which is formed in oxidative-reductive processes. Oxygenases may be monooxygenases (hydroxylases) and dioxygenases. They oxidize substrates by dint of inclusion of oxygen.

The structure and catalytic role of Pyridine enzymes

Pyridine enzymes consist of apoenzyme and coenzyme as NAD or NADP. NAD is nicotinamide adenine dinucleotide. NADP is nicotinamide adenine dinucleotide phosphate. Both of them consist of adenine, 2 riboses, 2 phosphates and nicotinamide (vitamin PP). NADP also contains additional phosphate.

NADP-dependent pyridine enzymes take part in synthetic processes (e.g. biosynthesis of free fatty acids etc):

S+NADPH2SH2+NADP

They occur in cytosol. NAD_dependent pyridine enzymes are contained in mitochondrias and take part in BO by dint of dehydrogenation of substrates. Hence they are primary DH-ases:

SH2+NADS+NADH2+energy

NADH2 are oxidized by FP (with FMN). Some substrates are oxidized by FP with coenzyme FAD.

The structure and catalytic role of Flavin enzymes (FP)

Flavin enzymes consist of apoenzyme and coenzyme as FMN and FAD. FMN is flavinmononucleotide (v.B2+H3PO4); FAD is flavinadeninedinucleotide (FMN + AMP).

Flavin ring carries out a catalytic role.

FP with FAD are primary DH-ases because they oxidize substrates, e.g. succinate dehydrogenase (SDH) oxidizes succinic acid to fumaric acid. FPH2 are oxidized by CoQ (Ubiquinone). Some FP, e.g. Xanthine oxidase etc may be oxidized by molecular oxygen, i.e. they possess the autooxidability: FPH2 + O2 = FP + H2O2

The structure and catalytic role of Ubiquinone

Coenzyme Q is so named because its apoenzyme hasn’t been found. The term “Ubiquinone” is from “ubiquity” – it occurs in all types of cells:

Reduced ubiquionone is oxidized without any enzymes. Hydrogen atoms are just broken up on protons and electrons. Protons are used in formation of endogenic water. Electrons are carried on cytochromes

The structure, catalytic role and representatives of Cytochromes

Cytochromes are two-component enzymes. They consist of apoenzyme and coenzyme as heme. Cytochromes are divided into 4 groups – A, B, C, D. each group is subdivided into subgroups, so group A includes Cyta and Cyta3; group B includes Cytb and Cytb5; group C includes Cytc and Cytc1. Cytochromes of group D occur in plants. Cytochromes of each group differ from each other by structure of heme. Cytochromes of subgroups differ from each other by structure of apoenzyme, i.e. they contain the same hemes.

Cytochromes of group B have got a heme which looks like heme of hemoglobin and myoglobin:

1,3,5,8-tetramethyl-2,4-divinyl-6,7-dipropionic acid iron porphin

The heme of cytochromes of group C is: 1,3,5,8-tetramethyl-2,4-diethyl-6,7-dipropionic acid iron porphin

The heme of cytochromes of group A is: 1,3-dimethyl-2-radical-4-vinyl-8-formyl-6,7-dipropionic acid iron porphin

The iron of hemes of cytochromes may be 3+ and 2+:

+e

Cyt (Fe3+) -------------------------------------------- Cyt (Fe2+)

(ferri) -------------------------------------------- (ferro)

– e

ferri form of cytochrome is oxidized. Ferro form of cytochrome is reduced one.

The subsequence of cytochromes in BO depend on their oxidative-reductive potential. They are located according increase of their potential: cytb cytc1cytccytacyta3O2

The structure and catalytic role of Catalase and peroxidases

Catalase and peroxidases consist of apoenzyme and coenzyme as heme. Apoenzyme is represented by 1 polypeptide chain. Heme is the same of heme of hemoglobin and myoglobin but iron has 3 valency. Catalase contains 4 hemes whereas peroxidases contain 1 heme.

Catalase catalyzes the next reaction: 2H2O2  2H2O + O2

Peroxidases catalyze the reaction: H2O2  H2O + [O]

The structure and catalytic role of oxygenases

Oxygenases consist of apoenzyme and coenzyme as vitamin C (ascorbic acid).

MOG oxidize substrates by dint of inclusion of atom of oxygen. This results in formation of hydroxylated product or product like SO: S+O2+NADPH2SO(H)+H2O+NADP

DOG include molecule of oxygen in substrate. This results in cleavage of double bond:

S+O2SO2 (e.g. formation of vitamin A from carotene and formation of dioxide of oleinic acid).