Enzymes: Structure, Function, and Regulation - A Comprehensive Overview | Study notes Biochemistry

Charles Andrew B. Martin

Enzymes

• Enzymes are catalysts and are not consumed

in the reactions

• They are proteins that act as a catalyst for

biochemical reactions

• The human body has 1000s of enzymes

• Enzymes are the most effective catalysts

known

• Most enzymes are globular proteins

• A few enzymes are now known to be ribonucleic

acids (RNA)

• Enzymes undergo all the reactions of proteins

including denaturation

• Enzyme activity is dramatically affected by:

o Alterations in pH

o Temperature

o Other protein denaturants

Enzyme Structure

Simple and Conjugated Enzymes

• Enzymes are of two types: simple enzymes

and conjugated enzymes

• Simple enzyme: composed only of protein

(amino acid chains)

• Conjugated enzyme: Has a nonprotein part

in addition to a protein part.

o Apoenzyme: Protein part of a

conjugated enzyme.

o A cofactor: Nonprotein part of a

conjugated enzyme.

o A holoenzyme is the biochemically

active conjugated enzyme

o Apoenzyme + cofactor = holoenzyme

(conjugated enzyme)

Cofactors

• Cofactors are important for the chemically

reactive enzymes

• Cofactors are small organic molecules or

inorganic ions

o Organic molecule cofactors: also

called as co-enzymes or co-

substrates

o Co-enzymes/co-substrates are

derived from dietary vitamins

• Inorganic ion cofactors

o Typical metal ion cofactors - Zn2+,

Mg2+, Mn2+, and Fe2+

o Non-metallic ion cofactor - Cl

o Inorganic ion cofactors derived from

dietary minerals

Terms in Enzyme Understanding

• Apoenzyme: the protein part of an enzyme.

• Cofactor: a nonprotein portion of an enzyme

that is necessary for catalytic function;

examples are metallic ions such as Zn2+ and

Mg2+.

• Coenzyme: a nonprotein organic molecule,

frequently a B vitamin, that acts as a cofactor.

• Substrate: the compound or compounds

whose reaction an enzyme catalyzes.

• Active site: the specific portion of the enzyme

to which a substrate binds during reaction

• Activation: any process that initiates or

increases the activity of an enzyme.

• Inhibition: any process that makes an active

enzyme less active or inactive.

• Competitive inhibitor: any substance that

binds to the active site of an enzyme thereby

preventing binding of substrate.

• Noncompetitive inhibitor: any substance that

binds to a portion of the enzyme other than the

active site and thereby inhibits the activity of the

enzyme.

Nomenclature and Classification of Enzymes

• Nomenclature: Most commonly named with

reference to their function

o Type of reaction catalyzed

o Identity of the substrate

• A substrate is the reactant in an enzyme-

catalyzed reaction:

o The substrate is the substance upon

which the enzyme "acts."

o E. g., In the fermentation process sugar

to be converted to CO2, therefore in this

reaction sugar is the substrate.

Three Important Aspects of the Naming Process

1. Suffix -ase identifies it as an enzyme

o E.g., urease, sucrase, and lipase are all

enzyme designations

o Exception: The suffix -in is still found in the

names of some digestive enzymes, E.g.,

trypsin, chymotrypsin, and pepsin

2. Type of reaction catalyzed by an enzyme is

often used as a prefix

o E.g., Oxidase - catalyzes an oxidation

reaction,

o E.g., Hydrolase - catalyzes a hydrolysis

reaction

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Enzymes

Enzymes are catalysts and are not consumed in the reactions
They are proteins that act as a catalyst for biochemical reactions
The human body has 1000s of enzymes
Enzymes are the most effective catalysts known
Most enzymes are globular proteins
A few enzymes are now known to be ribonucleic acids (RNA)
Enzymes undergo all the reactions of proteins including denaturation
Enzyme activity is dramatically affected by: o Alterations in pH o Temperature o Other protein denaturants Enzyme Structure Simple and Conjugated Enzymes
- Enzymes are of two types: simple enzymes and conjugated enzymes
- Simple enzyme : composed only of protein (amino acid chains)
- Conjugated enzyme : Has a nonprotein part in addition to a protein part. o Apoenzyme : Protein part of a conjugated enzyme. o A cofactor : Nonprotein part of a conjugated enzyme. o A holoenzyme is the biochemically active conjugated enzyme o Apoenzyme + cofactor = holoenzyme (conjugated enzyme) Cofactors
- Cofactors are important for the chemically reactive enzymes
- Cofactors are small organic molecules or inorganic ions o Organic molecule cofactors : also called as co-enzymes or co- substrates o Co-enzymes/co-substrates are derived from dietary vitamins
- Inorganic ion cofactors o Typical metal ion cofactors - Zn2+, Mg2+, Mn2+, and Fe2+ o Non-metallic ion cofactor - Cl o Inorganic ion cofactors derived from dietary minerals Terms in Enzyme Understanding
Apoenzyme : the protein part of an enzyme.
Cofactor : a nonprotein portion of an enzyme that is necessary for catalytic function; examples are metallic ions such as Zn2+^ and Mg2+.
Coenzyme : a nonprotein organic molecule, frequently a B vitamin, that acts as a cofactor.
Substrate : the compound or compounds whose reaction an enzyme catalyzes.
Active site : the specific portion of the enzyme to which a substrate binds during reaction
Activation : any process that initiates or increases the activity of an enzyme.
Inhibition : any process that makes an active enzyme less active or inactive.
Competitive inhibitor : any substance that binds to the active site of an enzyme thereby preventing binding of substrate.
Noncompetitive inhibitor : any substance that binds to a portion of the enzyme other than the active site and thereby inhibits the activity of the enzyme. Nomenclature and Classification of Enzymes
Nomenclature: Most commonly named with reference to their function o Type of reaction catalyzed o Identity of the substrate
A substrate is the reactant in an enzyme- catalyzed reaction: o The substrate is the substance upon which the enzyme "acts." o E. g., In the fermentation process sugar to be converted to CO 2 , therefore in this reaction sugar is the substrate. **Three Important Aspects of the Naming Process

Suffix -** ase identifies it as an enzyme o E.g., urease, sucrase, and lipase are all enzyme designations o Exception: The suffix - in is still found in the names of some digestive enzymes, E.g., trypsin, chymotrypsin, and pepsin 2. Type of reaction catalyzed by an enzyme is often used as a prefix o E.g., Oxidase - catalyzes an oxidation reaction, o E.g., Hydrolase - catalyzes a hydrolysis reaction

3. Identity of substrate is often used in addition to the type of reaction o E.g. Glucose oxidase, pyruvate carboxylase, and succinate dehydrogenase Practice Exercise Predict the function of the following enzymes. a. Maltase b. Lactate dehydrogenase c. Fructose oxidase d. Maleate isomerase Answers: a. Hydrolysis of maltose; b. Removal of hydrogen from lactate ion; c. Oxidation of fructose; d. Rearrangement (isomerization) of maleate ion Six Major Classes

Enzymes are grouped into six major classes based on the types of reactions they catalyze Classes Reaction Catalyzed

Oxidoreductases Oxidation-reductions
Transferases Functional group transfer reactions
Hydrolases Hydrolysis reactions
Lyases Reactions involving addition or removal of groups
Isomerase Isomerization reactions
Ligases Reactions involving bond formation coupled with ATP hydrolysis Oxidoreductase

An oxidoreductase enzyme catalyzes an oxidation-reduction reaction : o Oxidation and reduction reactions are always linked to one another o An oxidoreductase requires a coenzyme that is either oxidized or reduced as the substrate in the reaction o E.g., Lactate dehydrogenase is an oxidoreductase and the reaction catalyzed is shown below Transferase
A transferase is an enzyme that catalyzes the transfer of a functional group from one molecule to another (Ex. Aspartate amino transferase)
Two major subtypes: o Transaminases - catalyze transfer of an amino group to a substrate o Kinases - catalyze transfer of a phosphate group from adenosine triphosphate (ATP) to a substrate Hydrolase
A hydrolase is an enzyme that catalyzes a hydrolysis reaction (Ex. Acetylcholinesterase)
The reaction involves addition of a water molecule to a bond to cause bond breakage
Hydrolysis reactions are central to the process of digestion: o Carbohydrases hydrolyze glycosidic bonds in oligo- and polysaccharides (see reaction below) o Proteases effect the breaking of peptide linkages in proteins, o Lipases effect the breaking of ester linkages in triacylglycerols Lyase
A lyase is an enzyme that catalyzes the addition of a group to a double bond or the removal of a group to form a double bond in a manner that does not involve hydrolysis or oxidation (Ex. Aconitase)

Forces That Determine Substrate Binding

H-bonding
Hydrophobic interactions
Electrostatic interactions Enzyme Specificity 1. Absolute Specificity o An enzyme will catalyze a particular reaction for only one substrate o This is most restrictive of all specificities (not common) o E.g., Urease is an enzyme with absolute specificity
1. Stereochemical Specificity o An enzyme can distinguish between stereoisomers. o Chirality is inherent in an active site (amino acids are chiral compounds) o L-Amino-acid oxidase - catalyzes reactions of L-amino acids but not of D-amino acids. 3. Group Specificity o Involves structurally similar compounds that have the same functional groups. o E.g., Carboxypeptidase: Cleaves amino acids one at a time from the carboxyl end of the peptide chain 4. Linkage Specificity o Involves a particular type of bond irrespective of the structural features in the vicinity of the bond o Considered most general of enzyme specificities o E.g., Phosphatases: Hydrolyze phosphate- ester bonds in all types of phosphate esters Factors that Affect Enzyme Activity 1. Temperature
- Higher temperature results in higher kinetic energy which causes an increase in number of reactants collisions, therefore there is higher activity.
- Optimum temperature: The temperature at which an enzyme exhibits maximum activity
- Optimum temperature for human enzymes is 37°C (body temperature) - Increased temperature (high fever) leads to decreased enzyme activity 2. pH
pH changes affect enzyme activity
Drastic changes in pH can result in denaturation of proteins
Optimum pH: pH at which enzyme has maximum activity
Most enzymes have optimal activity in the pH range of 7.0 - 7. o Exception: Digestive enzymes o Pepsin: Optimum pH = 2. o Trypsin: Optimum pH = 8. 3. Substrate Concentration
Substrate Concentration : At a constant enzyme concentration, the enzyme activity increases with increased substrate concentration.
Substrate saturation : the concentration at which it reaches its maximum rate and all of the active sites are full
Turnover Number : Number of substrate molecules converted to product per second per enzyme molecule under conditions of optimum temperature and pH

Enzyme Concentration

Enzymes are not consumed in the reactions they catalyze
At a constant substrate concentration, enzyme activity increases with increase in enzyme concentration o The greater the enzyme concentration, the greater the reaction rate Practice Exercise
Describe the effect that each of the following changes would have on the rate of a reaction that involves the substrate sucrose and the intestinal enzyme sucrase. a) Decreasing the sucrase concentration b) Increasing the sucrose concentration c) Lowering the temperature to 10°C d) Raising the pH from 6.0 to 8.0 when the optimum pH is 6. Answers: a. Decrease rate b. Increase rate c. Decrease rate d. Decrease rate Enzyme Inhibition
Enzyme Inhibitor : a substance that slows down or stops the normal catalytic function of an enzyme by binding to it.
Competitive Inhibitors : Compete with the substrate for the same active site o Will have similar charge & shape o Noncompetitive Inhibitors: Do not compete with the substrate for the same active site o Binds to the enzyme at a location other than active site Enzyme Kinetics in the presence/absence of Inhibitors Reversible Competitive Inhibition
A competitive enzyme inhibitor: resembles an enzyme substrate in shape and charge
Binds reversibly to an enzyme active site and the inhibitor remains unchanged (no reaction occurs)
The enzyme - inhibitor complex formation is via weak interactions (hydrogen bonds, etc.).
Competitive inhibition can be reduced by simply increasing the concentration of the substrate. Reversible Noncompetitive Inhibition
A noncompetitive enzyme inhibitor decreases enzyme activity by binding to a site on an enzyme other than the active site.
Causes a change in the structure of the enzyme and prevents enzyme activity.
Increasing the concentration of substrate does not completely overcome inhibition. o Examples: Heavy metal ions Pb+, Ag*, and Hg2+. Irreversible Inhibition
An irreversible enzyme inhibitor inactivates enzymes by forming a strong covalent bond with the enzyme's active site. o The structure is not similar to enzyme's normal substrate o The inhibitor bonds strongly and increasing substrate concentration does not reverse the inhibition process o Enzyme is permanently inactivated. o E.g., Chemical warfare agents (nerve gases) and organophosphate insecticides

Antibiotics That Inhibit Enzyme Activity

An antibiotic is a substance that kills bacteria or inhibits their growth
Antibiotics usually inhibit specific enzymes essential to life processes of bacteria
Two families of antibiotics considered in this discussion are sulfa drugs and penicillin. Sulfa Drugs
Gerhard Domagk - discovered antibacterial activity in sulfanilamide
First antibiotics in the medical field
Many derivatives of sulfanilamide collectively called sulfa drugs exhibit antibiotic activities
Sulfanilamide is structurally similar to PABA (p- aminobenzoic acid)
Many bacteria need PABA to produce coenzyme, folic acid
Sulfanilamide is a competitive inhibitor of enzymes responsible for converting PABA to folic acid in bacteria
Folic acid deficiency retards bacterial growth and that eventually kills them
Sulfa drugs don't affect humans because we absorb folic acid from our diet Penicillin
Accidently discovered by Alexander Fleming in 1928
Several naturally occurring penicillin and numerous synthetic derivatives have been produced
All have structures containing a four-membered Beta- lactam ring fused with a five-membered thiazolidine ring
Selectively inhibits transpeptidase by covalent modification of serine residue
Transpeptidase catalyzes the formation of peptide cross links between polysaccharides strands in bacterial cell walls Cipro
The antibiotic ciprofloxacin hydrochloride (Cipro for short)
Considered the best broad-spectrum antibiotics because it is effective against skin and bone infections as well as against infections involving the urinary, gastrointestinal, and respiratory systems
It is the drug of choice for treatment of traveler's diarrhea
Bacteria are slow to acquire resistance to Cipro.
- Biochemical threats associated with terrorism has thrust Cipro into the spotlight because it is effective against anthrax. Medical Uses of Enzymes
Diagnose certain diseases: o Enzymes produced in certain organ/tissues if found in blood may indicate certain damage to that organ/tissue General Characteristic of Vitamins
Organic compounds
Must be obtained from dietary sources
Human body can't synthesize in enough amounts
Essential for proper functioning of the body
Needed in micro and milligram quantities
1 Gram of vitamin B is sufficient for 500, people
Enough vitamin can be obtained from balanced diet
Supplemental vitamins may be needed after illness
Many enzymes contain vitamins as part of their structures – conjugated enzymes
Two Classes: o Water Soluble and Fat Soluble
Synthetic and natural vitamins are the same o 13 known vitamins Water-Soluble Vitamins Vitamin C
Humans, monkeys, apes and guinea pigs need dietary vitamins
Co-substrate in the formation of structural protein collagen
Involved in metabolism of certain amino acids
100 mg/day saturates all body tissues - Excess vitamin is excreted
RDA (mg/day): o Great Britain: 30 o United S t a t e s a n d Canada: 6 0 o Germany: 75 Vitamin B
The preferred and alternative names for the B vitamins
Thiamin (vitamin B,)
Riboflavin (vitamin B2)

Niacin (nicotinic acid, nicotinamide, vitamin B3)
Vitamin B. (pyridoxine, pyridoxal, pyridoxamine)
Folate (folic acid)
Vitamin B12 (cobalamin)
Pantothenic acid (vitamin B5)
Biotin
Exhibit structural diversity
Major function: B Vitamins are components of coenzymes Fat-Soluble Vitamins Vitamins A, D, E, K
Involved in plasma membrane processes
More hydrocarbon-like with fewer functional groups Vitamin A (Retinol)
Has role in vision - only 1/1000 of vitamin A is in retina
3 Forms of vitamin A are active in the body
Derived from β-carotene
Preformed vitamin A forms are called retinoids. The retinoids include retinal, retinol, and retinoic acid Functions of Vitamin A
Vision : In the eye- vitamin A combines with opsin protein to form the visual pigment rhodopsin which further converts light energy into nerve impulses that are sent to the brain.
Regulating Cell Differentiation - process in which immature cells change to specialized cells with function. o Examples: Differentiation of bone marrow cells white blood cells and red blood cell.
Maintenance of the health of epithelial tissues via epithelial tissue differentiation. o Lack of vitamin A causes such surfaces to become drier and header than normal harder than normal.
Reproduction and Growth : In men, vitamin A participates in sperm development. In women, normal fetal development during pregnancy requires vitamin A. Vitamin D (Calciferol)
Two forms active in the body: Vitamin D2 and D 3
Sunshine Vitamin: Synthesized by UV light from sun ( cholesterol is turned into Vitamin D )
It controls correct ratio of Ca and P for bone mineralization (hardening)
As a hormone it promotes Ca and P absorption in intestine Vitamin E (Tocopherol) - Four forms of Vitamin Es: a-, b-, g- and d-Vitamin E - α-tocopherol is the most active biological active form of Vitamin E - Peanut oils, green and leafy vegetables and whole grain products are the sources of vitamin E - Primary function: Antioxidant - protects against oxidation of other compounds Vitamin K (Coagulation) - Two major forms; K 1 and K 2 - K, found in dark green, leafy vegetables - K2 is synthesized by bacteria that grow in colon - Dietary need supply: ~1/2 synthesized by bacteria and 1/2 obtained from diet - Active in the formation of proteins involved in regulating blood clotting Additional Information - Enzyme : a biological catalyst. o With the exception of some RNAs that catalyze their own self-cleavage, all enzymes are proteins. o Enzymes can increase the rate of a reaction by a factor of 10^9 to 10^20 over an uncatalyzed reaction. o Some catalyze the reaction of only one compound. o Others are stereospecific; for example, enzymes that catalyze the reactions of only L-amino acids. o Others catalyze reactions of specific types of compounds or bonds; for example, trypsin that catalyzes hydrolysis of peptide bonds formed by the carboxyl groups of Lys and Arg. Catalytic Power: - Enzymes provide an alternative pathway for reaction, one with a significantly lower activation energy and, therefore, a faster rate.

Enzymes: Structure, Function, and Regulation - A Comprehensive Overview, Study notes of Biochemistry

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