topic 11 - Enzymes

Question 1

what is an Enzyme?

Answer 1

Class of protein (mostly but also some RNAs = ribozymes)

Question 2

what does an enzyme do?

Answer 2

Act as a catalyst for a biochemical reaction.

Question 3

is an Enzyme changed permanently when used as a catalyst?

Answer 3

Not permanently changed in the reaction

Sometimes it is changed temporarily.

Question 4

what is the catalytic power of an Enzyme?

Answer 4

Enzymes do not invent new reactions

Enzymes do not change possibility of reaction to occur (energetics)

Enzymes increase the rate of reaction by factor of 1011 or higher.

Question 5

what is the rate of Enzymes?

Answer 5

H2O2 is the substrate and H2O + O2 are the products as hydrogen peroxide is a powerful oxidising agent and therefore needs to be detoxified rapidly.

A single molecule of catalase can break 5.6 million molecules of hydrogen peroxide each minute.

Question 6

what is the prevalence of Enzymes?

Answer 6

In an animal cells there are normally 1000 – 4000 different enzymes

Some are found in the majority of cells as they synthesis common cellular products e.g. proteins, nucleic acids, phospholipids or energy production

Others are present only a particular cell type e.g. nerve e.g. acetlycholinesterase.

Question 7

what does the name of an Enzyme usually indicate?

Answer 7

The name of an enzyme usually indicates its substrate and function e.g. alcohol dehydrogenase or glycogen phosphorylase.

Question 8

what does the suffix “ase” mean to enzymes?

Answer 8

The suffix –ase is often added to the name of the type of molecule on which an enzyme acts.

proteases – degrade proteins

phosphatases - remove phosphates,

ribonucleases - cleave RNA molecules.

Question 9

how do enzymes work?

Answer 9

They work by lowering the activation energy required for the molecular reaction to take place

In order to do so, an enzyme molecule must interact specifically - even if very briefly - with the substrate molecule or one or a small number of closely related substrates

The molecule (or substrate) fits within a particular cavity on the enzyme (active site) and is attracted to particular points of charge within the cavity (e.g. only L steroisomers of amino acids).

Question 10

what are the co-factors of an enzyme?

Answer 10

The catalytic activity of many enzymes depends on the presence of components called co-factors

Co-factors can be considered “helper molecules” that assist in biochemical transformations.

They can either be small molecules (organic) or metals (non organic).

They can also be classified depending on how tightly they bind to an enzyme, with loosely-bound cofactors termed coenzymes and tightly-bound cofactors termed prosthetic groups.

Question 11

what are prosthetic groups and co-enzymes?

Answer 11

Prosthetic small inorganic ions… mostly (vitamins) metal ions: Cu, Mg, Mn, Fe, Zn, Cu, Mg, Mn, Ni, Mo
- act as activators and/or inhibitors of activity

Co-enzymes
- small non-protein molecules that catalyse reactions
- Transfer electrons
- Form or break a covalent bond
- Transfer a group

Examples (metabolite and vitamin)
- NAD+/NADH
- Co-A
- Vitamins (Vitamin C, folic acid, B6 – pyridoxidal
phosphate)

Question 12

are binding sites specific in enzymes?

Answer 12

Substrate binding site is highly specific

‘Lock-and-key’ model: 3D shape recognizes substrate (hydrophobic, electrostatic, hydrogen bonds)

‘Induced-fit’ model: enzyme conformational change after binding substrate.

Hexokinase reacts strongly with glucose
Affinity for galactose is low
Galactokinase reacts strongly with galactose.

Question 13

what is the energy profile?

Answer 13

Activation energy: barrier to spontaneous reaction

Enzymes work by lowering the activation energy required for a reaction to proceed

Enzyme reduces the activation energy required to bring about the transformation of substrate to product.

Transition-state complex is stabilized by diverse interactions

Question 14

does pH and temperature affect enzymes?

Answer 14

Enzymes function within narrow ranges of pH and temperature.

Usually you find the enzyme in the appropriate microenvironment for its optimum functioning
- e.g. pepsin in stomach functions at low pH
- salivary amylase pH 6.8
- Taq polymerase for PCR at 72oC

Question 15

how does pH affect enzyme activity?

Answer 15

Each enzyme has characteristic pH optimum:
- depends on active site amino acids.
- depends on hydrogen bonds required for 3D structure.

Question 16

how does temperature affect enzyme activity?

Answer 16

An enzyme is a big molecule interacting with other molecules

Temperature causes the rate of interactions to go up; therefore the rate of catalysis goes up

HOWEVER, higher temperatures cause denaturation of enzyme

shape changes; therefore loses its activity.

Question 17

how do substrates affect enzyme activity?

Answer 17

1. At low concentration of substrate

Steep increase in the rate of reaction with increasing substrate concentration.

The catalytic site of the enzyme is empty, waiting for substrate to bind

The rate at which product can be formed is limited by the concentration of substrate which is available.

Question 18

how do substrates affect enzyme activity pt 2?

Answer 18

2) As the concentration of substrate increases

The enzyme becomes saturated with substrate

As soon as the catalytic site is empty, more substrate is available to bind.

The rate of formation of product now depends on the activity of the enzyme

Adding more substrate will not affect the rate of the reaction to any significant effect.

Question 19

what is the Michaelis-Menten kinetics equation?

Answer 19

Holds for an enzyme (E) combining with substrate (S) to form a complex (ES).

ES can break up to E + S…..or go on to form Product (P)…

Assuming the formation of P is irreversible.

      k1             k3 E + S --->   ES --------> EP
      <---
       k2

Question 20

what is the effect of amount of product on enzymes/ enzyme activity?

Answer 20

Amount of product has minimal effect on enzyme activity.

Question 21

what is enzyme inhibition?

Answer 21

An inhibitor binds to an enzyme and decreases its activity

Question 22

what is physiological innhibition? enzyme inhibition

Answer 22

Physiological inhibition

e.g. negative feedback to control a physiological process e.g. hexokinase by glucose 6-phosphate

inhibition by substances designed to treat disease.

Drugs

e.g. Allopurinol to treat Gout; inhibits Xanthine Oxidase which is involved in the production of uric acid

Poisons
e.g. Nerve gases inhibit acetlycholinesterase such as Sarin used in world war 2.

Question 23

what is competitive inhibition?

Answer 23

inhibitor competes reversibly with substrate for the active site.

Question 24

what is uncompetitive inhibition?

Answer 24

inhibitor binds only to the ES complex, leading to EIS intermediates. this is very rare.

Question 25

what is non competitive inhibition?

Answer 25

inhibitor binds non covalently to sites other than the active site (allosteric inhibition). Kinetics are complex and partially inhibited enzymes can still turn over substrate.

Question 26

what is irreversible inhibition?

Answer 26

irreversible inhibitors form covalent or very tight bonds with functional groups in active site.

Question 27

summary of enzymes

Answer 27

Enzymes are nature’s catalysts and are involved in nearly every biochemical process

They have an active site which is both highly specific for the substrates and positions them in such a way as to reduce the activation energy for the reaction

The products are not strongly bound, so are released, and the enzyme is recycled

Their kinetics are well described by the Michaelis-Menten model

Enzymes can be inhibited by other molecules

Binding to the active site

Binding elsewhere, resulting in a change of conformation

Reversibly or covalently in either case.