MEDICAL BIOCHEMISTRY:ENZYMES

MR. GENARO F. ALDERITE JR,MSERM

ENZYMES

- A protein with catalytic properties due to its power of specific activation

Characteristics of Enzymes1) biological catalysts

2) not consumed during a chemical reaction

3) speed up reactions from 1000 - 1017, with a mean increase in rate of 00,000

4) exhibit stereospecificity --> act on a single stereoisomer of a substrate

5) exhibit reaction specificity --> no waste or side reactions

Classification of Enzyme Specificity

a. Absolute specificity: substrate

Succinic dehydrogenase- succinic acid to fumaric acid

b. Linkage specificity:reaction that break bonds

Thrombin- acids arginine and glycine

c. Reaction specificity: reactions

Esterases- hydrolysis of esters

d. Group Specificity: compounds

chymotrypsin- catalyzes only protein that contains phenylalanine, tryptophan and tyrosine

Classification of Enzymes:1. According to its composition:

a. Simple enzymes-

b. Complex enzymes

holoenzyme - a complete, catalytically active enzyme including all co-factors

apoenzyme - the protein portion of a holoenzyme minus the co-factors

prosthetic group - a metal or other co-enzyme covalently bound to an enzyme

2. Class of organic chemical reaction catalyzed:

a. Oxidoreductase - catalyze redox reactions

*dehydrogenases, oxidases, peroxidases, reductases

Dehydrogenase-catalyze the removal of H from a substrate

Oxidases- activate oxygen so that it will readily c ombine with a substrate

b. Transferases - catalyze group transfer reactions; often require coenzymes

c. Hydrolases - catalyze hydrolysis reactions

  Carbohydrates

1. ptyalin- salivary amylase

-catalyze the hydrolysis of starch to dextrin and maltose

2. sucrase- hydrolysis of sucrose to glucosE and fructose

- intestinal juices

3. maltase- hydrolysis of maltose to glucose

4. Lactase- hydrolysis of lactose to glucose and

galactose

5. amylopsin- pancreatic amylase

- hydrolysis of starch to dextrins and maltose *from pancreas to Sintestine*

Esters- catalyze the hydrolysis of esters into acids and alcohol

1. Gastric lipase- hydrolysis of fats to fatty acids and alcohol

- part of the gastric juices

2. Steapsin- ( pancreatic lipase)

- hydrolysis of fats to fatty acids and glycerol

Proteases- catalyze the hydrolysis of derived proteins and amino acids

1. pepsin- hydrolysis of protein to polypeptides

2. trypsin- found in pancreatic juice

3. chymotrypsin

Hydrolysis Reaction

Lactose + H2O-galactosidase

Glucose + Galactose

d. Lyases - lysis of substrate; produce contains double bond

e. Isomerases - catalyze structural changes; isomerization

f. Ligases - ligation or joining of two substrates with input of energy, usually from ATP hydrolysis; often called synthetases or

synthases

Chemical reactions

• Chemical reactions need an initial input of energy = THE ACTIVATION ENERGY

• During this part of the reaction the molecules are said to be in a TRANSITION STATE

Reaction Pathway

Making Reactions Go Faster• Increasing the temperature make molecules move

faster

• Biological systems are very sensitive to temperature changes.

• Enzymes can increase the rate of reactions without increasing the temperature.

• They do this by lowering the activation energy.

• They create a new reaction pathway “a short cut”

An Enzyme Controlled Pathway

ENZYMATIC REACTION PRINCIPLES

• Biochemically, enzymes are highly specific for their substrates and generally catalyze only one type of reaction at rates thousands and millions times higher than non-enzymatic reactions. Two main principles to remember about enzymes are 1) they act as CATALYSTS (they are not consumed in a reaction and are regenerated to their starting state) and 2) they INCREASE THE RATE of a reaction towards equilibrium (ratio of substrate to product), but they do not determine the overall equilibrium of a reaction.

CATALYSTS • A catalyst is unaltered during the course of a reaction and

functions in both the forward and reverse directions. In a chemical reaction, a catalyst increases the rate at which the reaction reaches equilibrium, though it does not change the equilibrium ratio. For a reaction to proceed from starting material to product, the chemical transformations of bond-making and bond-breaking require a minimal threshold amount of energy, termed activation energy. Generally, a catalyst serves to lower the activation energy of a particular reaction.

ENZYMATIC REACTION PRINCIPLES (cont)

• The energy maxima at which the reaction has the potential for going in either direction is termed the transition state. In enzyme catalyzed reactions, the same chemical principles of activation energy and the free energy changes (Go) associated with catalysts can be applied. Recall that an overall negative Go indicates a favorable reaction equilibrium for product formation. As shown in an enzyme catalyzed reaction, and in the graph, the net effect of the enzyme is to lower the activation energy required for product formation.

Binding Energy

• The graph of activation energy and free energy changes for an enzymatic reaction also indicates the role binding energy plays in the overall process. Due to the high specificity most enzymes have for a particular substrate, the binding of the substrate to the enzyme through weak, non-covalent interactions is energetically favorable and is termed binding energy. The same forces important in stabilizing protein conformation (hydrogen bonding and hydrophobic, ionic and van der Waals interactions) are also involved in the stable binding of a substrate to an enzyme.

Reaction Rates• The rate of the reaction is determined by several factors

including:

A. The concentration of substrate

B. Temperature

C. pH.

A reaction rate will generally increase with increasing Temperature due to increased kinetic energy in the system untila maximal velocity is reached. Above this maximum, the kineticenergy of the system exceeds theenergy barrier for breaking weakH-bonds and hydrophobic interactions, thus leading tounfolding and denaturation of theenzyme and a decrease in reactionrate.

Effect of Temperature

Q10 (the temperature coefficient) = the increase in reaction rate with a 10°C rise in temperature.

For chemical reactions the Q10 = 2 to 3(the rate of the reaction doubles or triples with every 10°C rise in temperature)

Enzyme-controlled reactions follow this rule as they are chemical reactions

BUT at high temperatures proteins denature

The optimum temperature for an enzyme controlled reaction will be a balance between the Q10 and denaturation.

The effect of temperature

Temperature / °C

Enzyme activity

0 10 20 30 40 50

Q10 Denaturation

The effect of temperatureFor most enzymes the optimum temperature is about 30°C

Many are a lot lower, cold water fish will die at 30°C because their enzymes denature

A few bacteria have enzymes that can withstand very high temperatures up to 100°C

Most enzymes however are fully denatured at 70°C

Effect of pHVariations in pH can affect a particular enzyme in many ways, especially if ionizable amino acid side chains are involved in bindingof the substrate and/or catalysis. Extremes of pH can also lead todenaturation of an enzyme if the ionization state of amino acid(s) critical to correct folding are altered. The effects of pH and temperature will vary for different enzymes and must be determined experimentally.

Extreme pH levels will produce denaturationThe structure of the enzyme is changed

The active site is distorted and the substrate molecules will no longer fit in it

At pH values slightly different from the enzyme’s optimum value, small changes in the charges of the enzyme and it’s substrate molecules will occur

This change in ionisation will affect the binding of the substrate with the active site.

Optimum pH values

Enzyme activity Trypsin

Pepsin

pH1 3 5 7 9 11

Theories on Enzyme Specificity

1. The Lock and Key Hypothesis

2. The Induced Fit Hypothesis

The Lock and Key Hypothesis• Fit between the substrate and the active site of the

enzyme is exact • Like a key fits into a lock very precisely• The key is analogous to the enzyme and the

substrate analogous to the lock. • Temporary structure called the enzyme-substrate

complex formed • Products have a different shape from the substrate • Once formed, they are released from the active site • Leaving it free to become attached to another

substrate

Enzyme may be used again

Enzyme-substrate complex

E

S

P

E

E

P

Reaction coordinate

The Induced Fit Hypothesis

• Some proteins can change their shape (conformation)

• When a substrate combines with an enzyme, it induces a change in the enzyme’s conformation

• The active site is then moulded into a precise conformation

• Making the chemical environment suitable for the reaction

• The bonds of the substrate are stretched to make the reaction easier (lowers activation energy)

The Induced Fit Hypothesis

Hexokinase (a) without (b) with glucose substratehttp://www.biochem.arizona.edu/classes/bioc462/462a/NOTES/ENZYMES/enzyme_mechanism.html

Hexokinase Active Site: Glucose vs. Galactose Binding

INDUCED FIT

LOCK-AND-KEY

Catalytic Mechanisms: Types

• Four types of catalytic mechanisms will be discussed:

• binding energy catalysis

• general acid-base catalysis

• covalent catalysis

• metal ion catalysis

Many reactions involve the formation of normally unstable, chargedintermediates. These intermediates can be transiently stabilized in anenzyme active site by interaction of amino acid residues acting as weakacids (proton donors) or weak bases (proton acceptors). The general acid and general base forms of the most common and best characterizedamino acids involved in these reactions are shown above.

Acid-BaseCatalysis

Acid-Base Catalysis (cont)• The preceding functional groups can potentially serve as either

proton donors or proton acceptors. This is dependent on many factors including the molecular nature of the substrate, any co-factors involved, and the pH of the active site (which would determine the ionization state of an amino acid side chain). For acid-base catalysis, histidine is the most versatile amino acid due to its pKa which means that in most physiological situations it can act as either a proton donor or proton acceptor. Generally these amino acids will interact together with the substrate, or in conjunction with water or other weak, organic acids and bases found in cells.

Binding Energy Catalysis• Binding energy accounts for the overall lowering of activation energy for a

reaction, and it can also be considered as a catalytic mechanism for a reaction. Several catalytic factors in the binding of a substrate and enzyme can be considered: 1) transient limiting of substrate and enzyme movement by reducing the relative motion (or entropy) of the two molecules, 2) solvation disruption of the water shell is thermodynamically favorable, and 3) substrate and enzyme conformational changes. All three of these factors individually or in combination are utilized to some degree by an enzyme. While in some instances these forces alone can account for catalysis, they are frequently components of a complex catalytic process involving factors discussed for the other types of catalytic mechanisms.

Covalent Catalysis

• This mechanism involves the transient covalent binding of the substrate to an amino acid residue in the active site. Generally this is to the hydroxyl group of a serine, although the side chains of threonine, cysteine, histidine, arginine and lysine can also be involved.

Metal Ion Catalysis

• Various metals, all positively charged and including zinc, iron, magnesium, manganese and copper, are known to form complexes with different enzymes or substrates. This metal-substrate-enzyme complex can aid in the orientation of the substrate in the active site, and metals are known to mediate oxidation-reduction reactions by reversible changes in their oxidation states (like Fe3+ to Fe2+).

Summary of Catalytic Mechanisms

• In general, more than one type of catalytic mechanism will occur for a particular enzyme via various combinations of binding energy, acid-base, covalent and metal catalysis. Enzymes as a whole are incredibly diverse in their structures and the types of reactions that they catalyze, therefore there is also a large diversity of catalytic mechanisms utilized, the basis of which must be determined experimentally.

Inhibitors

• Inhibitors are chemicals that reduce the rate of enzymic reactions.

• The are usually specific and they work at low concentrations.

• They block the enzyme but they do not usually destroy it.

• Many drugs and poisons are inhibitors of enzymes in the nervous system.

The effect of enzyme inhibition

• Irreversible inhibitors: Combine with the functional groups of the amino acids in the active site, irreversibly.

Examples: nerve gases and pesticides, containing organophosphorus, combine with serine residues in the enzyme acetylcholine esterase.

Reversible inhibitors: These can be washed out of the solution of enzyme by dialysis.

Two Categories:

1.Competitive: These compete with the substrate molecules for the active site.

The inhibitor’s action is proportional to its concentration.

Resembles the substrate’s structure closely.

2. Non-competitive: These are not influenced by the concentration of the substrate. It inhibits by binding irreversibly to the enzyme but not at the active site.

Examples

• Cyanide combines with the Iron in the enzymes cytochrome oxidase.

• Heavy metals, Ag or Hg, combine with –SH groups.

T hese can be removed by using a chelating agent such as EDTA.

Medicine inhibitors:a. Methotrexate in cancer chemotherapy to semi-selectively inhibit DNA synthesis of malignant cells

b. Aspirin to inhibit the synthesis of prostaglandins which are at least partly responsible for the aches and pains of arthritis

c. Sulfa drugs to inhibit the folic acid synthesis that is essential for the metabolism and growth of disease-causing bacteria

Activators: are molecules that increase activity.

Examples:

Lipases- Used to assist in the removal of fatty and oily stains.

Amylases Detergents- for machine dish washing to remove resistant starch residues.

Papaine- To soften meat for cooking.

Clinical Use of Enzymes• Enzyme Activity in Body Fluids Reflects Organ Status:

• Cells die and release intracellular contents; increased serum activity of an enzyme can be correlated with quantity or severity of damaged tissues (ex. creatine kinase levels following heart attack)

• Increased enzyme synthesis can be induced and release in serum correlates with degree of stimulation (ex. alkaline phosphatase activity as a liver status marker)

Clinical Use of Enzymes (cont)

• Enzyme Activity Reflects the Presence of Inhibitors or Activators

• Activity of serum enzymes decreases in presence of an inhibitor (ex. some insecticides inhibit serum cholinesterases)

• Determine co-factor deficiencies (like an essential vitamin) by enzyme activity (ex. add back vitamin to assay, if activity increases, suggests deficiency in that vitamin)

Clinical Use of Enzymes (cont)• Enzyme activity can be altered genetically• A mutation in an enzyme can alter its substrate affinity, co-factor

binding stability etc. which can be used as a diagnostic in comparison with normal enzyme

• Loss of enzyme presence due to genetic mutation as detected by increased enzyme substrate and/or lack of product leading to a dysfunction

• NOTE: PCR techniques that identify specific messenger RNA or DNA sequences are replacing many traditional enzymatic based markers of genetic disease

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 .

Commonly assayed enzymes :a.amino transferases: b. alanine transaminase, ALT (sometimes still referred to as serum glutamate-pyruvate aminotransferase, SGPT) c. aspartate aminotransferase, AST (also referred to as serum glutamate-oxaloacetate aminotransferase, SGOT); d. lactate dehydrogenase, LDH; e. creatine kinase, CK (also called creatine phosphokinase, CPK);f. gamma-glutamyl transpeptidase, GGT.

-The typical liver enzymes measured are AST (aspartate aminotransferase), and ALT(Alanine transaminase) .

-Normally in liver disease or damage that is not of viral origin the ratio of ALT/AST is less than 1. However, with viral hepatitis the ALT/AST ratio will be greater than 1.

The 5 types and their normal distribution and levels in non-disease/injury are listed below. (lactate dehydrogenase )

• LDH 1 – Found in heart and red-blood cells and is 17% – 27% of the normal serum total.

• LDH 2 – Found in heart and red-blood cells and is 27% – 37% of the normal serum total.

• LDH 3 – Found in a variety of organs and is 18% – 25% of the normal serum total.

• LDH 4 – Found in a variety of organs and is 3% – 8% of the normal serum total.

• LDH 5 – Found in liver and skeletal muscle and is 0% – 5% of the normal serum total.

• CPK( Creatine phosphokinase) 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 significant amounts in smooth muscle and is 0% of the normal serum total.