An Introduction to Enzyme Structure and Function

An Introduction to Enzyme Structure and Function

Enzymes Many reactions in living systems are similar to laboratory reactions. 1. Reactions in living systems often occur with the aid of enzymes. 2. Enzymes are proteins produced by living systems which catalyze specific biological reactions. 3. ther requirements: vitamins, minerals, or hormones in order to catalyze a specific reaction.

Enzymes Enzymes are roteins Enzymes possess unique catalytic properties which depend upon their structural integrity. 1. artial hydrolysis of an enzyme results in the loss of enzymatic activity. 2. Activity is also lost under conditions that denature enzymes: high temperature and extremes of ph.

Enzymes Enzymes are roteins Catalysis takes place at active sites on the enzyme s surface. 1. An active site is a cleft or indentation occupying a very small part of the enzyme s surface. 2. art of the active site s structure provides the catalytic ability of the enzyme. 3. art of the active site s structure functions as a binding site for the substrates of the reaction being catalyzed.

Enzymes Active Site Enzyme Enzyme-Substrate Complex

Enzymes Enzymes are Catalysts A catalyst lowers the activation energy of a reaction by providing a different pathway leading to products.

What is a Catalyst Reactants roducts uncatalyzed reaction otential energy Ea Ea catalyzed reaction Reactants roducts Reaction coordinate

Enzymes Enzymes are Catalysts A catalyst lowers the activation energy of a reaction by providing a different pathway leading to products. It does so by becoming an active participant in the chemical process, but it emerges unchanged. It does not alter the results of a reaction, it changes only the speed at which the reaction takes place.

Enzymes are Superior Catalysts 1. Catalytic power - Most biochemical reactions would occur too slowly without a catalyst. 2. Specificity - The selectivity of enzymes towards the thousands of different possible substrates that exist in a cell is very strong. Specificity can be toward a specific compound, a specific type of chemical bond, or even a specific stereoisomer. 3. Enzymes can be regulated by a biological response. 4. Enzymes function within physiological constraints. a. ~ 37º c. Aqueous system b. ~ ph 7.3-7.5 d. Limited reagents

Enzyme Substrate Interactions Enzyme-substrate attractions takes place via non-covalent intermolecular forces. Enzyme Surface Substrate Molecule

Enzyme Substrate Interactions The enzyme-substrate interaction at the active site is sometimes called a lock and key fit (a). (The substrate is complementary to the active site in both shape and stereochemistry.)

Enzyme Substrate Interactions In most cases, the enzyme changes upon substrate binding This phenomenon is called inducible fit (b). (The enzyme changes the complementary character of the binding site as it binds the substrate.)

Inducible-Fit Model

Enzyme Cofactors and Coenzymes Most enzymes are combined with other small chemical entities called cofactors or coenzymes. The protein portion alone is called the apoenzyme. The protein portion together with its cofactors and coenzymes is called the holoenzyme. ne purpose of cofactors and coenzymes is to maintain the protein portion of the enzyme in the correct conformation. Cofactors are usually metal cations. Coenzymes are usually organic in nature.

Enzyme Cofactors and Coenzymes apoenzyme cofactors or coenzymes active enzyme

Enzyme Cofactors and Coenzymes Many catalytic functions cannot be accomplished using the functional groups provided by the amino acid side chains of a protein alone. In these cases, coenzymes carrying the necessary organic functional groups, are transiently bound to the apoenzyme. These coenzymes are usually derived from vitamins.

Enzyme Classification Classification according to SUBSTRATE (the specific compound upon which an enzyme acts) SUBSTRATE(S) Enzyme RDUCT(S) Examples: Galactosidase, Ribonuclease, Urease, Lipase, eptidase, Esterase, Amidase Classification according to TYE F REACTIN CATALYZED 1. XIDREDUCTASES 2. TRANSFERASES 3. HYDRLASES 4. ISMERASES 5. LYASES - Catalyze addition or 6. LIGASES - Catalyze formation of removal of small molecules bonds using the energy from AT hydrolysis

fficial Classes of Enzymes

fficial Classes of Enzymes

fficial Classes of Enzymes 1. xidoreductases Alcohol dehydrogenase (EC 1.1.1.1) (oxidation with NAD+) NAD+ NADH/H+ H 3 C H C H H H 3 C C H Ethanol Acetaldehyde 2. Transferases Hexokinase (EC 2.7.1.2) (phosphorylation) CH 2 H AT AD CH 2 3 2- H H H H H H H H D-Glucose D-Glucose-6-phosphate

fficial Classes of Enzymes 3. Hydrolases Carboxypeptidase A (EC 3.4.17.1) (peptide bond cleavage) H 2 N R' C C N R C C N R' C C + H 3 N R C C H H H H H H H C-terminus of polypeptide Shortened polypeptide C-terminal residue 4. Lyases yruvate decarboxylase (EC 4.1.1.1) (decarboxylation) C C yruvate CH 3 + H+ + C H C CH 3 Acetaldehyde

fficial Classes of Enzymes 5. Isomerases Maleate isomerase (EC 5.2.1.1) (cis-trans isomerization) C H C C C C C C H C H H Maleate Fumarate 6. Ligases yruvate carboxylase (EC 6.4.1.1) (carboxylation) C C CH 3 yruvate + C AT AD + i C C C CH 2 xaloacetate

Enzyme Activity If a fixed amount of enzyme is present (as is the case in most biological systems) the rate of an enzyme reaction can be measured as a function of the substrate concentration. The rate of the reaction reaches a maximum value as the substrate concentration is increased. At this point the enzyme is said to be saturated. When saturated with substrate, the enzyme activity is described as the turnover number of the enzyme: the number of substrate molecules converted into product molecules per enzyme molecule per unit time. Enzymes, may increase the rate of a reaction over that of the uncatalyzed reaction by factors up to 100,000,000.

Enzymes are Superior Catalysts Enzyme Reaction Turnover Number molecules/sec

Enzyme Activity If an excess of substrate is present (as is the case in most clinical laboratory test systems) the initial rate of an enzyme reaction can be measured as a function of the enzyme concentration. The rate of an enzyme catalyzed reaction also increases with an increase in the enzyme concentration. In the presence of excess substrate, the observed enzyme activity (per unit time) is proportional to the amount of enzyme present. This factor can be used in medical diagnosis of cell damage where cell contents leak from damaged cells.

Enzyme Activity : Temperature and ph What factors influence the 3-D conformation of an enzyme? Reaction Rate vs Temperature Reaction Rate vs ph

Enzyme Activity Blood levels of enzymes following heart attack Troponins Relative amount above normal Blood levels of troponins, creatine phosphokinase (CK-2), aspartate transaminase (AST), and lactate dehydrogenase (LDH) in the days following a heart attack.

Control of Enzyme Activity

Control of Enzyme Activity Which factors influence whether an enzyme reaction is taking place: 1. Is the enzyme present? 2. Is the substrate or cofactor present? 3. Is the enzyme in its correct 3-D conformation? 4. Is the active site of the enzyme vacant and ready to accept a substrate molecule?

Control of Enzyme Activity Is the enzyme present? 1. Enzymes are synthesized under the direction of a gene (a particular segment of DNA) a. Repressors - Bind to DNA and prevent protein synthesis b. Inducers - Cause a change in the repressor molecule which drops off DNA 2. Enzymes may be synthesized in an inactive form: RENZYME (zymogen) --------------> ACTIVE ENZYME

Control of Enzyme Activity Are the substrate or necessary cofactors present? 1. Another enzyme might synthesize the substrate or cofactor. Enzyme 1 Enzyme 2 Enzyme 3 A B C D 2. Access of the substrate or cofactor to the enzyme may be limited by membrane permeability. 3. A necessary cofactor or precursor may not be present in the diet. (vitamin deficiency)

Control of Enzyme Activity What factors influence the 3-D conformation of an enzyme? Reaction Rate vs Temperature Reaction Rate vs ph

Biochemical Control Mechanisms Feedback Inhibition A molecule at the end of a biosynthetic pathway inhibits an enzyme at the beginning of the pathway. Enzyme 1 Enzyme 2 Enzyme 3 A B C D

Reversible Enzyme Inhibitors Inhibitors are molecules that affect activity of an enzyme towards its substrate. Competitive inhibitors: resemble the substrate but do not undergo a reaction. They bind to the active site and exclude the substrate. The binding is reversible and the inhibitor can be displaced by raising the substrate concentration. Uncompetitive inhibitor Uncompetitive inhibitors: are not structurally related to the substrate. They bind to a site on the enzyme distinct from the active site. Raising the substrate concentration does not reverse this type of inhibition.

Kinetics of Enzyme Inhibitors

Noncompetitive or Uncompetitive Inhibition inhibitor binds to enzyme-substrate complex, altering the enzyme s conformation enzyme-substrate complex forms enzyme-product complex releases product at a reduced rate or, in some cases, the inhibitor prevents the binding of a second substrate

Allosteric Control Mechanisms Many regulatory enzymes of metabolic activity are allosteric enzymes. The activity of an allosteric enzyme is controlled by the binding of specific molecules called activators and inhibitors. The activator or inhibitor binds to a site on the enzyme distinct from the substrate binding site. Binding of the activator or inhibitor results in a conformational change of the enzyme which either increases or decreases its catalytic activity.

Irreversible Enzyme Inhibitors Essentially oisons Alter the enzyme through a permanent covalent modification An example - The nerve gas Sarin inactivates the enzyme acetylcholine esterase. H 3 C CH 3 CH F CH 3 Sarin

roposed Mechanism for Acetylcholine Esterase Acetylcholine H 3 C H 3 C CH 3 CH 2 N CH 2 C CH 3 Acetic Acid H CH 3 C C + Glu H Ser H 3 C CH 3 CH 2 N CH 2 H Acetylcholine Esterase H 3 C Choline

roposed Mechanism for Nerve Gas Sarin C Glu H 3 C CH 3 CH F H CH 3 Sarin H 3 C Acetylcholine CH 3 CH 2 N CH 2 C CH 3 Ser H 3 C X Acetylcholine Esterase C Glu H CH 3 C 3 CH CH 3 + HF Ser Covalent Bond Irreversibly Inhibits Enzyme

Control of Enzyme Activity Which factors influence whether an enzyme reaction is taking place: 1. Is the enzyme present? 2. Is the substrate or cofactor present? 3. Is the enzyme in its correct 3-D conformation? 4. Is the active site of the enzyme vacant and ready to accept a substrate molecule?

Biochemical Control Mechanisms: Covalent Modification: Reversible phosphorylation and dephosphorylation. ENZYME Ser The Tyr ENZYME Ser The Tyr ENZYME ENZYME The modified enzyme (protein) may have increased or decreased biological activity

Covalent Modification of ne Enzyme by Another Many enzymes can exist in two forms, which are interconverted by phosphorylation and dephosphorylation. This covalent modification creates an enzyme with a different overall conformation. hosphorylation Ser Ser ENZYME Thr ENZYME Thr Tyr Tyr Dephosphorylation

A- Covalent Modification of ne Enzyme by Another The source of the phosphate groups is usually AT. A- A- A- A- A- Ser Ser ENZYME Thr ENZYME Thr Tyr Tyr

Covalent Modification of ne Enzyme by Another A- Specific enzymes are needed to catalyze the transformations. A- A- A- A- A- rotein Kinase Ser Ser ENZYME Thr ENZYME Thr Tyr Tyr rotein hosphatase

+ Covalent Modification of ne Enzyme by Another By turning protein kinases and protein phosphatases on and off reciprocally, a cell choses which form of the enzyme will be present. + _ rotein Kinase Ser Ser ENZYME Thr ENZYME Thr Tyr Tyr rotein hosphatase _

Covalent Modification of ne Enzyme by Another rotein Kinase Ser Ser ENZYME Thr ENZYME Thr Tyr Tyr Sometimes it is the nonphosphorylated form of the target enzyme which is active. rotein hosphatase So where is the regulation? Sometimes it is the phosphorylated form of the target enzyme which is active.

Entire hysiological Responses Can Be Controlled In This Manner

hysiological Response rotein Kinase 1 rotein Kinase 2 hysiological Response

hysiological Response rotein hosphatase A hysiological Response rotein hosphatase B