Enzymes 4 - Harmer

Question 1

Cofactors are important in enzymes because

Answer 1

non-amino components of enzymes such as metals allow the enzymes access to chemistry that standard amino acids cannot provide

they increase their catalytic range

e.g. redox, carbon transfers, point charges

Question 2

There are two types of cofactors:

Answer 2

metal ions and organic cofactors

Question 3

Metal ions

Answer 3

Bound freely
Na, K, Mg, Ca, Zn, Cu, Fe, Mn

Bound as part of larger groups
Fe, Cu, Mn
e.g. Haemoglobin, iron sulfur clusters (to gather electrons and transfer them, they are really good at absorbing and storing single electrons, often found in protein arms to allow transfer of e from one place to another)

Question 4

Advantages of metal ions (4)

Answer 4

• structural stability: maintaining structure of enzyme
• target binding: unique properties
• catalysis: non protein catalysis
• redox: Fe and Cu especially can alter redox state, allows complex chemistry, also storage of electrons

matrix enzymes often use metal ions to help them fold

Question 5

Metalloproteases interact with water to

Answer 5

increase its nucleophilic properties

e.g. zinc can bind water, a nearby amino acid (often glutamate) acts as a BASE and deprotonates the oxygen to create OH- to hydrolyse the peptide bond in the substrate

Question 6

Non metal cofactors

Answer 6

e.g. NAD transfers a H+ and H-
e.g. biotin
e.g. thiamine diphosphate
e.g. cobalamin
generally contain large unique side groups to bind specific proteins

Question 7

Biotin mechanism - carboxylation

Answer 7

Biotin is covalently attached to the enzyme through an amide linkage to an active site lysine.

First, the bicarbonate ion is phosphorylated by ATP, and thus is activated for decarboxylation, which generates free CO2

Biotin’s job is to hold on to the carbon dioxide molecule until pyruvate comes into the active site. Carboxylation of biotin involves deprotonation of the amide nitrogen to form an enolate-like intermediate (amides have a pKa of approximately 17, and this is lowered by the presence of an active site acid near the oxygen).

This step is followed by attack of the nucleophilic nitrogen on carbon dioxide to form carboxybiotinylated enzyme

When a pyruvate molecule binds, rearrangement of the active site architecture causes the previous step to go in reverse, freeing the CO2 and generating a biotin base to deprotonate the alpha-carbon of pyruvate so that it can condense, in an aldol-like fashion, with CO2 to form oxaloacetate

Question 8

Thiamine diphosphate chemistry

Answer 8

the ring with N and NH2 allows H bond donors/acceptors easy loss/gain of protons

the middle ring with N+ and S stabilises the carbanion that can act as a nucleophile

the diphosphate group gives specificity and energy of binding

natural amino acids cannot make C-!!

Question 9

Cobalamin radical chemistry

Answer 9

uses cobalt!

carbon cobalt bond breaks homolytically (1 e each) to generate a carbon radical

the cofactor stabilises the radical so it is not damaging

carbon radical removes a proton from glutamic acid creating a glutamic acid radical in the active site

reaction transfers e to glycine creating a glycine radical

Question 10

Carboxylic acid reductase

Answer 10

use a 3 cofactor system to reduce RCOOH to RCHO (very unfavourable energetically)

ATP provides the energy

NADPH provides reducing power

phosphopantethiene arm switches the substrate between the two sites