Enzymes and coenzymes

Enzymes basic facts

Biological catalysts

• increase the rate of reaction by lowering activation energy

• Nearly all proteins, some are RNA (riboenzymes)

• Active site = area of specific AA residues

• Many have -are suffix

7 major classes of enzyme

1. Oxidoreductase

• transfer of electrons in redox reactions E.g. dehydrogenases

2. Transferase

• transfer function groups E.g. RNA polymerase

3. Hydrolase

• hydrolysis - water added E.g. proteases, ATPases

4. Lyase (Synthase)

• group removal, cleave bonds by elimination to form new products E.g. decarboxylase

5. Isomerase

• rearrangement of atoms in molecules E.g. cis-trans isomerases

6. Ligase (Synthetases)

• joining two molecules, needs energy E.g. DNA Ligase

7. Translocases

• movement of ions or molecules across membranes E.g. pumps

Examples of industrial/clinical uses

• Subtilisin = extracellular serine endopeptidase, biological action of washing powders

• Taq DNA Polymerase = used in PCR for amplification of DNA

• Calf rennet = mix of proteases, separates milk curds and whey

• Liver function tests = Alanine aminotransferase (ALT)

Coenzymes and Cofactors

• small non-protein molecule required for enzyme fun

Cofactors = inorganic ions e.g. Fe2+

Coenzymes = organic molecule e.g. ATP, FAD

Cosubstrates = loosely bound type of coenzyme, bind and unbind during catalysis

Prosthetic group = covalently bound cofactors/enzyme

Holoenzyme = catalytically active with its cofactor/enzyme

Apoenzyme = inactive, only protein

Basic thermodynamics

Cells, tissues and organs = systems

Entropy (S) = degree of randomness/disorder

2nd law of thermodynamics = change in entropy of system + change of entropy in surroundings = 0

Difficult to measure in biochemical processes

Gibbs Free Energy (G)

How do enzymes lower activation energy

• Enzymes lower the activation energy (change in GFE)

• Enzymes bind tightly to X (transitions state) not S or P

S→X→P

GFE of activation

A measure of the energy barrier that must be overcome for the reaction to proceed at a certain rate

Components of a holoenzyme

Enzyme catalytic strategies

Covalent catalysis

• Active site contains reactive groups, which covalently bonds to the substrate during the reaction

General acid-based catalysis

• transfer of protons (donate or accept) to or from and intermediate

Catalysis by approximation

• binding surface of enzyme brings two substrates into close proximity so a reaction can happen

Metal ion catalysis

• ions can act as an electrophile (electron acceptor) and stabilise negative charge

• Facilitate nucleophile formation by coordination

• Act as a bridge between enzyme and substrate

Proenzymes/Zymogens

• Enzymes synthesised as larger inactive precursors

• Zymogens activated by proteolytic (hydrolytic) removal of a peptide

• E.g. trypsin ‘cuts’ lots of enzymes to activate them

• Require a biochemical change to activate

Substrate specificity of endopeptidases

Endopeptidases → split peptide bonds in proteins creating smaller proteins

Catalytic triad of Chymotripsin

Covalent catalysis→ impermanent covalent bond between enzyme and substrate

• Histidine 57 is a proton acceptor (nucleophile)

• Pulls H+ from O-H bond of Serine 195

• Carboxyl group of Aspartate hydrogen bonds with histidine to orient it to be a proton acceptor

• Two phase process

Chymotripsin → Acetylation

• Aspartate 102 helps orient Histidine, which then acts as a base to pull a H from Serine’s alcohol group

• Activated Serine attacks the carbonyl carbon of the peptide bonds on the substrate

• This forms a temporary, unstable ‘tetrahedral intermediate’

• Intermediate collapses, Histidine donates a H+ which breaks the peptide bond and releases the C-terminal portion of the peptide

• The N-terminal portion stays covalently bonded to Serine

Chymotripsin → Deacylation

• A water molecule enters the active site

• Histidine removes a H from the water molecule, making it a strong nucleophile

• Activated water molecule attacks the carbonyl carbon on the Acyl-enzyme

• A second intermediate collapses, Histidine donates its proton back to Serine, breaks bond between enzyme and substrate

• N terminal released and active site regenerates