biochem exam enzymes

enzymes

Almost all are globular proteins

• Act as catalysts for biological reactions

– Work by lowering the energy of activation (energy

needed to cause or allow a chemical reaction)

• They are not destroyed during the reaction

– However, they undergo same reactions as proteins,

including hydrolysis (degradation/digestion) and

denaturation

• Most are switched on or off by substrate/product

concentration and hormones (regulation)

enzyme specificity

Enzymes vary in their degree of specificity

(binding) for substrates

– Some are highly specific (bind with only one

substrate; i.e., glucose)

– Others are moderately specific (bind with

molecules that are chemically similar; i.e.,

hexoses)

– Others are not very specific (bind a large

number of substrates; i.e., phosphate

containing)

• Specificity can often be seen in their names

6 classes of enzymes

Oxidoreductases: oxidation/reduction reactions

(one substrate gets oxidized while the other

substrate gets reduced

– Dehydrogenases

• Transferases: transfer of a functional group

from 1 substrate to other substrate

– Transaminases, kinases

• Hydrolases: hydrolysis reactions (break bonds

of H2O (substrate) to add to other substrate)

– Lipases, proteases, digestive enzymes

Lyases: removal of a group of atoms (not O

or H) from one substrate

– Decarboxylases, deaminases

• Isomerases: change or move a functional

group within a single substrate

– Isomerases, mutases

• Ligases: catalyze the bonding together of two

substrates to form one larger product

– Synthases

enzyme structure

Simple enzymes: an enzyme composed

only of protein (amino acids)

• Conjugated enzymes: an enzyme whose

structure has a non-protein (prosthetic)

group in addition to a protein portion

– Protein portion = apoenzyme

– Prosthetic group = cofactor or coenzyme

Cofactors

Usually metal ions (Co, Cu, Mg, Mn, and

Zn) (minerals in nutrition) that bind

transiently to the enzyme to enhance

substrate binding

– metalloenzymes

Coenzymes

Serve as recyclable shuttles or group

transfer agents

– Changed during the reaction

– Sometimes stated as a substrate

• Usually made from various B vitamins

– Thiamine (TPP), riboflavin (FAD, FMN),

niacin (NAD+, NADP+), pantothenic acid

(coenzyme A), B6 (PLP), biotin, folate (THF),

B12 (cobalamin), alpha lipoic acid

How do enzymes work?

Active site: relatively small part of an enzyme

that the substrate binds reversibly to during

reaction

• Substrate/reactant

– Forms an “enzyme-substrate complex”

– Chemical reaction occurs to convert substrate to

product

– Product is then released from active site

• Enzyme + substrate → ES → enzyme (recycled) +

product

Lock and Key Model

The active site has a fixed, rigid

geometrical shape

• Only a substrate with an corresponding

matching shape can fit into it

– i.e., how a key with a certain 3D shape fits

into a matching lock

Induced Fit Model

The active site changes its shape as the

substrate approaches to promote substrate

binding

– Thus, active site is not rigid but conforms to the

substrate shape for increase binding

• Cofactors are usually involved in helping the

substrate to bind to the active site

enzymes in diagnosing diseases

Enzymes are usually present in the blood in

very small amounts

• During disease states, cells die and lyse and

release enzymes into the interstitial fluid which

are picked up by the lymphatic system and

eventually seen in the blood

• Speed of enzyme rising in the blood is

proportional to its molecular weight (size)

– Smaller faster; larger slower

Clinically important enzymes

Amylase

– Used to diagnose acute pancreatitis

• Lipase

– Used to monitor recovery from acute pancreatitis

and/or diagnose chronic pancreatitis

• Acid phosphatase (acidphos or ACP)

– Prostatic acid phosphatase (PAP) is increased in

some stages of prostatic cancer

• Alkaline phosphatase (alkphos or ALP)

– Increases in bone disorders

Creatine kinase (CK)

– Increases first in myocardial infarction (MI) and

muscle wasting diseases

• Aspartate transaminase (AST, GOT, SGOT)

– Increases second in MI and liver diseases (liver

function assay)

• Alanine transaminase (ALT, GPT, SGPT)

– Liver diseases (liver function assay)

• Lactate dehydrogenase (LDH)

– Usually increased in many disease states (late in

MI, skeletal muscle and liver disease)

Isozymes

Multiple forms of the same enzyme

• They catalyze the same reaction but in

different tissues and have slightly different

amino acid compositions and sequence

– Example: 5 different structural forms of LDH

(liver vs heart vs skeletal muscle) but perform the

same biochemical reaction

– Because they contain a slightly different amino

acid composition, each isomeric form can be

separated and identified by electrophoresis

enzymes activity

Depends upon how easily the substrate can bind

to the enzyme

– Cofactors help this

• Kinetic Theory

– How much energy is needed to cause the chemical

reaction to occur (energy of activation)

– Increase kinetic energy overcomes energy of

activation (barrier)

• Concentrations of substrates and enzymes

• Saturation of active site

types of chemical reactions

Reversible reactions

– A + B ↔ C + D

– These are all substrates since the arrow goes in

both directions

– Depending upon which substrates are in higher

concentrations determines the direction

• Irreversible reactions

– A + B → C + D

– A and B are substrates and C and D are

products since the arrow goes in one direction

– This is due to energetics, or C and D are

converted into something else, or leave the cell

Factors Aaffecting Enzyme Activity

Temperature

– Increase temp leads to increase kinetic energy

and increase collision frequency between

enzyme and substrate

– As temp increases from low to high, the # of

molecules whose kinetic energy are > energy of

activation increases (↑ activity)

– At very high temperatures, however, the

enzyme can denature (↓ activity)

pH (7 – 7.5 for most) (2 – 3 for some)

- Alterations in pH affect structure

(conformation) of the enzyme

- Conformational change in active site

may increase or decrease substrate

binding

Substrate and Enzyme Concentration

– When [S] is high, there are many molecules

with enough energy to collide with enzyme for

reaction to occur

– Double substrate and rate will increase 2x,

double both substrate and enzyme and rate will

increase 4x

• Reaction rate is proportional to concentration of

molecules until saturation

• Inside cells, enzyme concentration doesn’t usually

change that much but substrate concentration does

based on several factors (predominantly feasting,

fasting and physical activity)

Inhibitors

Competitive Inhibition

– A molecule that resembles the substrate

(chemically and structurally) and reversibly binds

to the active site in place of substrate

– Temporarily prevents substrate from binding to the

active site

• It is reversible since forces holding inhibitor to active

site are weak interactions

There is competition for the active site

between the substrate and inhibitor

• Increase [S] can overcome the inhibition

• Competitive inhibition = Vmax doesn’t

change but Km increases

– You will still reach Vmax but it will take increase

amounts of substrate (Km) to get there

Non-Competitive Inhibition

– A non-similar molecule that reversibly binds to a

site on an enzyme other than active site

• This site is called an allosteric site

– The binding changes the conformational shape of

the enzyme including the active site thus

preventing the substrate from binding

Inhibition is not overcome by increase [S]

• Noncompetitive inhibition = Km doesn’t

change but Vmax decreases

– You will never reach Vmax no matter how much

substrate is added

Irreversible Inhibition

– A molecule that forms a strong covalent bond (not

reversible) to the active site or allosteric site

• Permanently prevents substrate from binding to the

active site

• Permanent deactivation of enzyme

– Increase [S] does not overcome inhibition

– You need to stop taking the inhibitor and

synthesize new enzyme to re-start reaction

homeostasis/metabolism

Cells have the ability to respond to changes in

their environment

• Substrate concentrations want to be in a steady

state and remain relatively constant

(homeostasis)

• When concentrations increase, enzymes are

activated to reduce concentration; when

concentrations decrease, other enzymes are

activated to increase concentration (metabolism)

• Problems with homeostasis can cause significant

harm to health of organism

Enzyme Regulation

Synthesis and Repression

– Synthesis: substrates or structurally similar

compounds, can induce synthesis of certain

enzymes when needed

• Liver alcohol dehydrogenase

– Repression: excess product inhibits synthesis

of enzyme and may even induce degradation

of enzyme

• Both can be governed by DNA / gene regulation

and RNA synthesis since enzymes are proteins

Allosteric Regulation

– An enzyme with 2 or more protein chains

(quaternary proteins) and 2 kinds of binding sites

(active site and allosteric site)

• Both sites are distinct and can be on two different chains

or two different locations on the same chain

– Substances that bind at the allosteric site are called

effectors

– Works by 3 different mechanisms

Positive effectors: substrate accepted more

readily; increases enzyme activity and reaction

rate

• Negative effectors: substrate accepted less

readily; similar to non-competitive inhibition;

decrease reaction rate

Feedback Inhibition

– Process in which activation or inhibition of the

allosteric enzyme of a pathway is controlled by a

product of the reaction or pathway

– Regulators in this situation are commonly products

of reaction & hormones from outside the cell

Indicator Metabolites

– Metabolic end products are regulators

– In this case, allosteric enzyme does decrease its

own synthesis but does not do it through enzyme

inhibition but through a different mechanism

outside the metabolic pathway; i.e. decreased RNA

synthesis thus decreasing enzyme synthesis or

concentration

Second Messengers

– Special allosteric regulators whose production or

release is triggered by an external first messenger

(hormone or nerve impulse)

• Second messengers include cAMP, Ca2+, phosphatidyl

inositols (PIP2)

Enzyme Regulation: Zymogens

Sometimes called proenzymes

– An inactive, pre-synthesized precursor of an

enzyme

– To become active; individual amino acid or a small

peptide is either added to or removed from enzyme

structure

– Examples: pepsinogen to pepsin

fibrinogen to fibrin

• Removal of peptide fragment is by hydrolysis

Enzyme Regulation: Covalent Modification

he addition or release of other atoms to one

or more amino acids in the peptide (usually

reversible) to activate or inactivate an enzyme

• Example: phosphorylation (protein kinases) or

dephosphorylation (protein phosphatases)

(addition or removal of phosphate group from an

amino acid)