An Introduction To Enzymes: (BRIEF HISTORY)

1835 Jon Jakob Berzelius, a Swedish chemist who termed the enzyme chemical action catalytic.

In the 1850s Louis Pasteur concluded that fermentation of sugar into alcohol by yeast is catalyzed by ferments. He postulated that these ferments, later named ENZYMES, are inseparable from the structure of living yeast cells. 1897 Eduard Buchner discovered that yeast extracts can ferment sugar to alcohol proved that the enzymes involved in fermentation can function when removed from the structure of living cells. 1926 James Sumners isolation and crystallization of urease provided a breakthrough in early studies of the properties of different enzymes. He found out that urease crystals consisted entirely of protein and postulated that all enzymes are proteins. Late 1930s after John Northrop and his colleagues crystallized pepsin and trypsin and found them also to be proteins, was Sumners conclusions widely accepted. During this period, J.B.S. Haldane wrote a treaties entitled ENZYMES. ENZYMES Highly specialized proteins Protein molecules that increase the rates of chemical reactions w/o themselves undergoing any change Reaction catalysts of biological systems Complex organic catalysts of highly specific action that are of vital importance in biological processes

As catalysts, ENZYMES are remarkable in 2 respects: 1. 2. They are extremely effective, increasing reaction rates by anywhere from 109 to 1020 times They are extremely specific

Four Distinct Types of Specificity

Absolute Specificity the enzyme will catalyze only on one reaction. Group Specificity the enzyme will act only on molecules that have specific functional groups. Linkage Specificity the enzyme will act on particular type of chemical bond regardless of the rest of the molecular structure. Stereochemical Specificity the enzyme will act on a particular steric or optical isomer.

NATURE OF ENZYMES Enzymes are proteins of low molecular weight. They are coagulated and so become in active by high temperature alcohol, salts of heavy metals, and alkaloidal reagents. In general, they are soluble in water. Most reactions of living matter are accelerated by enzymes. Enzymes are responsible for the reactions like oxidation, reduction, hydrolysis, synthesis and many others which are necessary during digestion, metabolism, respiration, energy transfers and release. References: Principle of Biochemistry, 2nd Edition By: Lehninger, Nelson, Cox Biochemistry, 3rd Edition

By: Mathews, Van Holde, Ahern Principles of Biochemistry By: Campbell

Enzymes are specialized proteins that function in the acceleration of chemical reactions. Catalysts increase the rate of a chemical reaction but is not itself change in the process. lowers the energy barrier to a reaction. 2 Important Characteristics of Enzyme 1. 2. Enzyme is not change as well as a result of catalysis. Enzyme does not change the equilibrium constant of the reaction but simply increases the rate at which the reaction approaches equilibrium.

Terminology:

Apoenzyme an inactive enzyme; is the protein part of an enzyme without any cofactors or prosthetic groups that may be required for the enzyme to be functional. Holoenzyme an active enzyme; it is the addition of a cofactor or prosthetic group to the apoprotein. Cofactors are small organic or inorganic molecules that an apoenzyme requires for its activity. Prosthetic Group is similar to a cofactor but is tightly bound to an apoenzyme. Substrate the molecule acted upon by the enzyme to form product. Substrate-binding Site a particular arrangement of amino acid side chains in the polypeptide that is specially formulated to bind a specific substrate; may contain the active site. Active Site a region of the enzyme where enzyme binds a molecule of substrate or in some cases several substrates. Isoenzymes (isozymes) enzymes that have variants that catalyze the same chemical reaction. Allosteric Site is a unique site where small molecules bind and effect a change in the substrate-binding site or the activity occurring in the active site.

TERMINOLOGY: Specificity refers to the ability of an enzyme to discriminate between two competing substrates. Catalysts lowers the energy barrier to a reaction. Michaelis Menten Equation the rate equation for one substance.

V=Vmax x [S]KM x [S] KMMichaelis constant Kcat Turnover number; measures the rate of catalytic process. Vmax Maximum initial velocity
Activation Energy energy input required to initiate the reaction.

Transition State an activated form of a molecule in which the molecule has undergone a partial chemical reaction; the highest point on the reaction coordinate. Ground State the normal, stable form of an atom or molecule; as distinct from the excited state. Standard Free Energy Change the free-energy change for a reaction occurring under a set of standard conditions. Substratescompound on which enzyme works, the one whose reaction it speeds up. Inhibitors a substrate that interferes with the action of an enzyme and slows the rate of a reaction. Coenzymes organic cofactors. Cofactors nonprotein portions of enzyme Allosteric Effector a substance that modifies the quaternary structure Concerted an allosteric enzyme can exist in an inactive Sequential there are still two conformation Cooperativity a phenomenon in biology displayed by enzymes or receptors that have multiple binding sites. Zymogen catalically inactive molecules; an example of proteolytic activation.

Vforward = Vreverse Vforward = K+1 [A] Vreverse = K-1 [B]

Enzymes in the Diagnosis of Pathology

The measurement of the serum levels of numerous enzymes has been shown to be of diagnostic significance. This is because the presence of these enzymes in the serum indicates that tissue or cellular damage has occurred resulting in the release of intracellular components into the blood. Hence, when a physician indicates that he/she is going to assay for liver enzymes, the purpose is to ascertain the potential for liver cell damage. Commonly assayed enzymes are the amino transferases: alanine transaminase, ALT (sometimes still referred to as serum glutamate-pyruvate aminotransferase, SGPT) and aspartate aminotransferase, AST (also referred to as serum glutamate-oxaloacetate aminotransferase, SGOT); lactate dehydrogenase, LDH; creatine kinase, CK (also called creatine phosphokinase, CPK); gamma-glutamyl transpeptidase, GGT. Other enzymes are assayed under a variety of different clinical situations but they will not be covered here. The typical liver enzymes measured are AST and ALT. ALT is particularly diagnostic of liver involvement as this enzyme is found predominantly in hepatocytes. When assaying for both ALT and AST the ratio of the level of these two enzymes can also be diagnostic. Normally in liver disease or damage that is not of viral origin the ratio of ALT/AST is less than 1. However, with viral heapatitis the ALT/AST ratio will be greater than 1. Measurement of AST is useful not only for liver involvement but also for heart disease or damage. The level of AST elevation in the serum is directly proportional to the number of cells involved as well as on the time following injury that the AST assay was performed. Following injury, levels of AST rise within 8 hours and peak 24-36 hours later. Within 3-7 days the level of AST should return to pre-injury levels, provided a continuous insult is not present or further injury occurs. Although measurement of AST is not, in and itself, diagnostic for myocardial infarction, taken together with LDH and CK measurements (see below) the level of AST is useful for timing of the infarct. The measurement of LDH is especially diagnostic for myocardial infarction because this enzyme exist in 5 closely related, but slightly different forms (isozymes). The 5 types and their normal distribution and levels in non-disease/injury are listed below. LDH LDH LDH LDH LDH 1 2 3 4 5 Found Found Found Found Found in in in in in heart and red-blood cells and is 17%-27% of the normal serum total. heart and red-blood cells and is 27%-37% of the serum total. a variety of organs and is 18%-25% of the normal serum total. a variety of organs and is 3%-8% of the normal serum total. liver and skeletal muscle and is 0%-5% of the normal serum total.

Following a myocardial infarct the serum levels of LDH rise within 24-48 hours reaching a peak by 2-3 days and return to normal in 5-10 days. Especially diagnostic is a comparison of the LDH-1/LDH-2 ratio. Normally, this ration is less than 1. A reversal of this ration is referred to as a flipped LDH. Following an acute myocardial infarct the flipped LDH ratio will appear in 12-24 hours and is definitely present by 48 hours in over 80% of patients. Also important is the fact that persons suffering chest pain due to angina only will not likely have altered LDH levels. CPK is found primarily in heart and skeletal muscle as well as the brain. Therefore, measurement of serum CPK levels is a good diagnostic for injury to these tissues. The levels of CPK will rise within 6 hours of injury and peak by around 18

hours. If the injury is not persistent the level of CK returns to normal within 2-3 days. Like LDH, there are tissue-specific isozymes of CPK and there designations are described below. CPK3 (CPK-MM) is the predominant isozyme in muscle and is 100% of the normal serum total. CPK2 (CPK-MB) accounts for about 35% of the CPK activity in cardiac muscle, but less than 5% in skeletal muscle and is 0% of the normal serum total. CPK1 (CPK-BB) is the characteristic isozyme in brain and is in normal serum total. Since most of the released CPK after a myocardial infarction is CPK-MB, an increased ratio of CPK-MB to total CPK may help in diagnosis of an acute infarction, but an increased of total CPK in itself may not. CPK-MB levels rise 3-6 hours after a myocardial infarct and peak 12-24 hours later if no further damage occurs and returns to normal 12-48 hours after the infarct.

Enzymes Assays Useful in Medical Diagnosis

Enzymes Alanine aminotransferase Acid Phosphatase Alkaline Phosphatase Aspartate Aminotransferase Lactate dehydrogenase Creatine Kinase Phosphohexose Isomerase Amylase

Normal Activity

Body Fluid Serum

Disease Diagnosed Hepatitis Prostate Cancer Liver/Bone Disease Heart Attack/ Hapatitis Heart Attack Heart Attack Heart Attack Pancreatic disease/ Mumps

3-17 U/L

2.5-12 U/L 13-38 U/L 7-19 U/L 7-49 U/L 100-350 WU/mL 7-60 U/L 15-75 U/L 19-80 U/L

Serum Serum Serum Cerebrospinal fluid Serum Serum Serum Serum

U/L = international units per liter WU/L = wrobleski units per milliliter HOW ENZYMES ACT AS CATALYSTS

Lock-and-Key Hypothesis According to this model, the enzyme accommodates the specific substrate as a lock does its specific key. Proposed by the great German biochemist Emil Fischer in 1894. Fischers idea: What fits the enzyme active site best is a substrate molecule induced to take up a configuration approximating the transition state. Induced Fit Hypothesis Proposed by Daniel Koshland in 1958. states that an enzyme induces a bound substrate molecule to adapt a conformation resembling the transition state. 2 Specific Enzyme-Catalyzed Reactions

1. TRIOSE PHOSPHATE ISOMERASE

catalyzes the interconvention of glyceraldehydes-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) a reaction that is known to proceed via an enediol intermediate

Catalysis by triose phosphate isomerase involves promotion and

stabilization of an enediol intermediate.

1. SERINE PROTEASES
catalysis of peptide bond hydrolysis this includes trypsin and chymotypsin

CLASSIFICATION OF ENZYMES

Enzymes are classified according to the report of a Nomenclature Committee appointed by the International Union of Biochemistry (1984). This enzyme commission assigned each enzyme a recommended name and a 4-part distinguishing number. The Enzyme Commission (EC) number divides enzymes into six main groups according to the type of reaction catalyzed. Four numbers, main class, subclass, sub-subclass, and serial number, separated by periods, designated an enzyme.

(1) Oxidoreductase catalyzes redox reactions Oxidized A + reduced B reduced A + oxidized B eg. Alcohol dekydrogenase includes: dehydrogenases oxygenases (2) Transferase catalyze the transfer of a functional group (e.g. acyl-, alkyl- and glycosyl-) A X + B A + B X eg. Hexokinase glucose + ATP glucose 6-phosphate + ADP oxidases peroxidases

an enzyme that transfers a phosphate from ATP to another compound is called kinase includes: phosphatases transmethylases (3) Hydrolase transaminases

 involves hydrolytic reactions and their reversal. A-B + H2O A + B eg. Lactase lactose + H2O glucose + galactose

enzymes that remove phosphate are called phosphotases

includes: esterases lipases sucrose maltase (4) Lyase

involves elimination reactions in which a group of atoms is removed from the

glycosidases proteases amylase lactase

substrate.

C A

C B

+ A -B

eg. Aldolase fructose 1.6 biphosphate dihydroxyacetonephosphate + glyceraldehydes 3-phosphate

includes: decarboxylase decarboxylases (5) Isomerase catalyzes molecular isomerizations A C C B C B C B aldolases dehydratases

eg. UDP galactose 4-epimerase

UDP galactose UDP glucose includes: epimerases racemates intramolecular transferases cis-trans isomerases

(6) Ligases also known as synthetases forms a relatively small group of enzymes which involve the formation of a covalent bond joining two molecules together, coupled with the hydrolysis of a nucleoside triphosphate. ATP + A + B A B + ADP + Pi eg. DNA Ligase

includes: synthetase

carboxylases

ENZYMES

WHERE BOUND

SUBSTRATE

END PRODUCTS

HYDROLYTIC CARBOHYDRATES
Ptyalin or salivary amylase Amylopsin or pancreatic amylase Maltase Lactase Sucrase Saliva Pancreatic juice Intestinal juice Intestinal juice Intestinal juice Starch & glycogen Starch & glycogen Maltose Lactose Sucrose Maltose Maltose Glucose Glucose & galactose Glucose

ESTERASES OR LIPASES
Gastric lipase Steapsin or pancreatic Phosphatases tissues Gastric juice in the stomach Pancreatic juice Emulsified fats Fats Hexophosphate Fatty acid & glycerol Fatty acid & glycerol Hexose & H3PO4

PROTEASES
Pepsin Trypsin Chymtrysin Aminoplypoptidasos & carboxypeptidases Dipeptidases Gastric juice Pancreatic juice Pancreatic juice Intestinal juice Intestinal & Pancreatic juice Proteins Proteis, proteoses, peptones Proteis, proteoses, peptones Polypeptides Dipeptides Proteases & peptoxes Polypeptides Polypeptides Peptides & amino acids Amino acids

OXIDO-REDUCTASES
dehydrogenases Animal tissues Plant & animal tissues Hydrogen donors Carbohydrate, proteins & fats in tissues Oxidized hydrogen acceptor CO2 & H2O

Oxidases

Peroxidases Catalases

Plant & animal tissues Plant & animal tissues

Organic Peroxides Hydrogen Peroxides

Oxygen Water & oxygen

FERMENTING ENZYMES
Zymase Lactic acidase Yeast Lactic acid bacteria Monosaccharide Lactose CO2 ethyl alcohol Lactic acid

COAGULATING ENZYMES
Rennin Gastric juice Milk or casein Paracasein

Secretin-Chemical Messenger

Naming of enzymes Traditionally, enzymes were simply assigned names by the investigator who discovered the enzyme. A few enzymes discovered before the naming system was devised are known by common names.

Examples are pepsin, trypsin, and chymotrypsin which catalyzes the hydrolysis of proteins. As knpwledge expanded, systems of enzymes classification become more comprehensive and complex. The latest systematic nomenclature system known as the International Enzyme Commission (IEC) system is based upon the type of reaction catalyzed. Enzymes are commonly named by adding ase to the root name of substrate molecule it is known about what it does, or how much is desired to be communicated. Generally, it will include the type of reaction and/or the chemical that is reacting. The enzymes name is comprised of the names of the substrate(s), the product(s) and the enzymes functional class. Three names of enzymes Recommende name- convient form used in regular discourse ( lactate Dehydrogenase) Systematic name- more specific, less ambiguous, corresponds 1:1 with EC number(L-lactate: NAD oxidoreductase) Enzyme Commision ( EC) numeric designation(1.1, !.27)

Rules of Enzymes Systematic name should have TWO parts: a) The name of the substrate/s b) The processes specifying name endings in -ase Use trivial names of substrates, or even abbreviations when they have very widely accepted meaning Use the name of true substrate form Use hyphens to keep each substrates name to one unit Choose substrates for only one reaction direction consistently within the entire class of enzymes Specify the location of substituent with 1, 2 ,3 Phosphate containing compounds with no other acidic group are named using xxx-phosphate form With ketoacid class compounds, use the oxo prefix when-CO-replaces CHand the Keto prefix when CO-replaces a CHOH- in a sugar For nucleic radicals use adenylyl rather than adenyl