Enzymes may be defined as protein catalysts that catalyze chemical reaction in biological systems But this definition is not entirely correct, as same RNA molecules, called Ribozymes, have now been found to catalyze some reactions

As per this system, the name starts with EC (enzyme class) followed by 4 digits. First digit represents the class Second digit stands for the subclass Third digit is the sub-subclass or subgroup Fourth digit gives the number of the particular enzyme in the list.

Class 1: Oxidoreductases Class 2: Transferases Class 3: Hydrolases Class 4: Lyases Class 5: Isomerases Class 6: Ligases Enzyme The enzymes are grouped into following six major classes

Catalyse oxidation of one substrate with simultaneous reduction of another substrate or co-enzyme Example- Alcohol dehydrogenase Alcohol + NAD Aldehyde + NADH + H+

Transfers one group (other than hydrogen) from the substrate to another substrate Example- Hexokinase Hexose + ATP Hexose-6-phosphate + ADP

Hydrolyse ester, ether, peptide or glycosidic bonds by adding water & then breaking the bond Example- Acetyl choline esterase Acetyl choline + H2O Choline + acetate

Remove groups from substrates or break bonds by mechanisms other than hydrolysis Example- Aldolase Fructose-1,6-bisphosphate Glyceraldehyde- 3-phosphate +dihydroxy acetone phosphate

Produce optical, geometric or positional isomers of substrates Example- Triose phosphate isomerase Glyceraldehyde-3-phosphate Di-hydroxy- acetone-phosphate

Link two substrates together, usually with the simultaneous hydrolysis of ATP Example- Acetyl CoA carboxylase Acetyl CoA + CO2 + ATP Malonyl CoA + ADP +Pi

Enzymes are protein catalysts that increase the velocity of a chemical reaction and are not consumed during the reaction A. Active site- A. Active site- Active site of an enzyme represents as the small region at which the substrate binds and participates in the catalysis Substrate Active site Enzyme

 Existence is due to the tertiary structure of protein  Made up of amino acids known as catalytic residues  Regarded as cleft occupying a small region in a big enzyme molecule and not rigid in structure and shape  Generally, possesses a substrate binding site and a catalytic site  Substrate binds at the active site by weak non-covalent bonds

 Specific in their function due to the existence of active sites  Commonly found amino acids at the active sites are serine, aspartate, histidine, cysteine, lysine, arginine, glutamate, tyrosine etc. Catalytic efficiency B. Catalytic efficiency - Enzymes-catalysed reactions are highly efficient, proceeding from times faster than uncatalysed reactions

D. Holoenzymes, apoenzymes, cofactors and coenzymes-  The term holoenzyme refers to the active enzyme with its non-protein component, whereas the enzyme without its non-protein moiety is termed an apoenzyme and is inactive.  If the nonprotein moiety is a metal ion, such as Zn 2  or Fe 2 , it is called a cofactor. If it is a small organic molecule, it is termed as coenzyme.

Inactive Enzyme Active Enzyme Co-Enzyme APOENZYME HOLOENZYME  Coenzyme are only transiently associate with the enzyme are called cosubstrates.

 Co-substrates dissociate from the enzyme in an altered state (for example NAD  ).  If the coenzyme is permanently associated with the enzyme and returned to its original form, it is called a prosthetic group (for example FAD ).  Coenzymes commonly are derived from vitamins for example NAD  and FAD.

Coenzymes may be divided in two groups 1. First Group of Co-enzymes 2. Second Group of Co-enzymes Coenzymes Coenzymes

1. First Group of Co-enzymes- change occurring in the substrate is counter-balanced by the co-enzymes. e.g. NADP–NADPH, FAD-FADH2 and FMN-FMNH2. Nicotinamide Adenine Dinucleotide (NAD+)- co-enzyme synthesized from Nicotinamide, a member of vitamin B complex.

2. Second Group of Co-enzymes- co-enzymes take part in reactions transferring groups other than hydrogen. Most of them belong to vitamin B complex group. e.g. Adenosine triphosphate (ATP), Thiamine pyrophosphate (TPP), Biotin, Coenzyme-A (Co-A) etc. Adenosine Triphosphate (ATP)- considered to be the energy currency in the body due to high energy bonds.

Co-enzyme Group transferred Thiamine pyrophosphate (TPP) Hydroxy ethyl Pyridoxal phosphate (PLP) Amino group Biotin Carbon dioxide Coenzyme-A (Co-A) Acyl groups Tetra hydrofolate (FH4) One carbon groups Adenosine triphosphate (ATP) Phosphate Examples of co-enzymes

Cofactors- Cofactors- The cofactor is an inorganic ion (metal or non-metal) for example, Ca 2 , Mg 2 , Fe 2 , Zn 2  or Cu 2  most of the cofactors are loosely (non- Covalently) attached to the apoenzyme and are not consumed in the reaction.

Metal is tightly bound and cannot be removed without disrupting the apo-enzyme architecture. Example- C arbonic anhydrase The metal ions are associated but not bound to the enzyme and hence, can be removed without causing any denaturation or change in three-dimension structure of the enzyme Example- Kinases

Metal Enzyme containing the metal Zinc Carbonic anhydrase, carboxy peptidase, alcohol dehydrogenase Magnesium Hexokinase, phospho fructo kinase, enolase, glucose-6-phosphatase Manganese Phospho gluco mutase, hexokinase, enolase, glycosyl transferases Copper Tyrosinase, cytochrome oxidase, lysyl oxidase, superoxide dismutase Iron Cytochrome oxidase, catalase, peroxidase, xanthine oxidase Calcium Lecithinase, lipase Molybdenum Xanthine oxidase Metallo-enzymes

What do Metal ion do in Enzymes?  Metal ion stabilize the three-dimensional structure of the enzyme and hence, contribute to the final conformation required for the reaction.  Metal ion are mostly found at the active site and hence, help in interaction with the substrate.  Metals found in the enzymes are transition state elements and thus have multiple oxidation states.

E. Regulation- E. Regulation- Enzyme activity can be regulated i.e. increased or decreased, so that the rate of product formation responds to the needs of the cell. F. Location within the cell- F. Location within the cell- Many enzymes are localized in specific organelles within the cell. Such compartmentalization serves to isolate the reaction substrate or product from other competing reactions.

CYTOSOL  Glycolysis  PP pathway  Fatty acid synthesis LYSOSOME  Degradation of complex macromolecules MITOCHONDRIA  TCA cycle  Fatty acid oxidation  Oxidation of pyruvate NUCLEUS  DNA and RNA synthesis Intracellular Location of important biochemical pathways

Apoenzyme Coenzyme Prosthetic group Cofactor Nature Protein Organic, Organic or Inorganic Nonprotein inorganic Source Specific Vitamins or Vitamins, heme & Inorganic gene nucleotides inorganic elements elements Examples All enzymes NAD, FAD, FMN, iron-heme Mg 2 ,Ca , TPP, ATP, UTP Cu 2 , Mn 2  Attachment to - Loose Very tight Loose the apoenzyme (noncovalent) (covalent or noncovalent) Heat stability Labile Fairly stable Stable Very stable Size Largest Smaller Smaller Smallest Determine Yes No No No Specificity Differences between the components of holoenzyme system

The mechanism of enzyme action can be viewed from two different perspectives.  The first treats catalysis in terms of energy changes that occur during the reaction. That is, enzymes provide an alternate, energetically favorable reaction pathway different from the uncatalyzed reaction.  The second perspective describes how the active site chemically facilitates catalysis. Mechanism of enzyme action Mechanism of enzyme action

Virtually all chemical reactions have an energy barrier separating the reactants and the products. This barrier, called the activation energy (E a ), is the energy difference between that of the reactants and a high-energy intermediate the transition state (T*) which is formed during the conversion of reactant to product. A T* B A. Energy changes occurring during the reaction A. Energy changes occurring during the reaction

Activation energy- Activation energy- Virtually all chemical reactions have an energy barrier, separating the reactants and the products. This barrier is called the free energy of activation, is the energy difference between the energy of the reactant and high energy intermediates that occurs during the formation of product

Reaction with enzyme Reaction kinetics without enzyme Time Substrate Energy Product C- e nergy level of substrate D - energy level of product C to A- activation energy in the absence of enzyme C to B- activation energy in the presence of enzyme B to A- lowering to activation energy by enzyme C D A B

1. Transition-state stabilization- 1. Transition-state stabilization- The active site often acts as a flexible molecular template that binds the substrate and initiates its conversion to transition state. By stabilizing the transition state, the enzyme greatly increases the concentration of the reactive intermediate that can be converted to product and thus, accelerates the reaction. 2. Catalysis- 2. Catalysis- The active site can provide catalytic groups that enhance the probability that the transition state is formed. In some enzymes, these groups can participate in general acid-base catalysis in which amino acid residues provide or accept protons. B. Chemistry of active site B. Chemistry of active site

Example of reaction specificity Pyruvate dehydrogenase Lactate dehydrogenase Pyruvate carboxylase Transaminase Acetyl- CoA Pyruvate Lactate OxaloacetateAlanine

The formation of an enzyme-substrate complex (ES) is very crucial for the catalysis to occur and for the product formation. Enzyme catalysed reaction proceeds 10 6 to times faster than a non-catalysed reaction. This is mainly due to four processes- 1. Acid-base catalysis 2. Substrate strain 3. Covalent catalysis 4. Proximity catalysis Mechanism of enzyme catalysis Mechanism of enzyme catalysis

1. Acid-base catalysis 1. Acid-base catalysis - Role of acids and bases is quite important in enzymology. At the physiological pH, histidine is the most important amino acid, the protonated form of which functions as an acid and its corresponding conjugate as a base. The other acids are -OH group of tyrosine, -SH group of cysteine and  -amino group of lysine. The conjugates of these acids and carboxyl ions (COO  ) function as bases. Example- Ribonuclease

2. Substrate strain- 2. Substrate strain- When a substrate binds to the preformed active site, the enzyme induces a strain to the substrate. The strained substrate leads to the formation to the formation of product. During the course of strain induction, the energy level of substrate is raised, leading to a transition state. Example- Lysozyme

3. Covalent catalysis 3. Covalent catalysis - The negatively charged (nucleophilic) or positively charged (electrophilic) group is present at the active site of the enzyme. This group attacks the substrate that results in the covalent binding of the substrate to the enzymes. Example- Serine proteases, chymotrypsin, trypsin, thrombin etc.

4. Proximity catalysis 4. Proximity catalysis - Enzymes enhance reaction rates by decreasing entropy. When correctly positioned and bound on the enzyme surface, the substrates are strained to the transition state. This is referred to as the proximity effect. The reactants should come in close proximity to the enzyme, for appropriate catalysis to occur. The higher concentration of the substrate molecule, the greater will be the rate of the reaction.