Enzyme regulation, isozymes and multiprotein complexes

feedback inhibition and enzyme pathways

Final products are often allosteric inhibitors of enzymes early in the pathway.

When product concentrations are high, feedback inhibition acts to reduce the 'flux' of the pathway so that less products are made.

Targeting enzymes early on in the pathway prevents build-up of potentially toxic intermediates and reduces waste products.

aspartate transcarbamoylase (ATCase)

Catalyses the first committed step, and the rate limiting step of pyrimidine biosynthesis.

The enzyme reaction does not follow the conventional hyperbolic form of Michaelis-Menten kinetics. There is substrate cooperativity in binding and feedback inhibition.

2 states of the enzyme

T - low affinity for substrates, low activity

R - high affinity for substrates, high activity

ordered binding of the substrates 

1. Carbamyl phosphate binds causing small structural changes 

2. Aspartate binds causing major changes between T to the R state and activating the enzyme for catalysis

Allosteric regulation by CTP and ATP alters the enzyme activity. CTP feedback inhibition of the pathway.

isozymes 

within the genome can be multiple genes which encode enzymes which perform the same chemical reactions 

why does this happen?

additional levels of control over enzyme activity for different biological environments 

Lactate dehydrogenase (EC 1.1.1.27)

During exercise, when muscles exhaust the oxygen, pyruvate gets catalysed into lactic acid by LDH to supply the necessary ATP.

Different isozymes are composed of different LDH subunits and expressed differently in different tissues.

use of LDH isozymes analysis

to diagnose tissue damage - anaemia, pulmonary embolism, hepatitis, acute renal failure

LDH levels

LDH level overall can be used to identify various cancers. High LDH activity is associated with malignancies.

• LDH-1 levels compared to LDH-2 levels are high it could be a sign of haemolytic anaemia

• Red blood cells are broken down and release LDH-1 into the blood stream

zymogens/proenzymes

Digestive serine proteases (trypsin)

Inactive trypsinogen is produced in the pancreas and only activated in the small intestine by N-terminal cleavage through enteropeptidase digestion.

Subsequent conformational changes through N-terminus complete the formation of the active site oxyanion hole for activation.

Tryptophan synthase (EC 4.2.1.20)

1. Salmonella typhimurium X-ray crystallography structure

2. Steady-state and single turnover experiments failed to trap indole as an intermediate

3. Beta-C-170F variant blocks the channel resulting in accumulation of indole

why?

Hydrophobic tunnel between alpha and beta domains allows for diffusion of the intermediate between domains without release to the solvent.

why channel substrates between domains

• Protection of reactive/toxic intermediates from bulk solvent

• Segregation of intermediates from competing enzymatic transformations

• Increased catalytic efficiency

• Tighter control of metabolic flux

• Decreased diffusion of intermediates away from reaction sites

• Allosteric control between domains

 

lysosomal acid lipase deficiency (LAL-D)

• Wolman disease

Early onset in infancy, organ damage and typically fatal within 6 months

• Cholesterol ester storage disease (CESD)

Later onset more variable in symptoms
Autosomal recessive mutations in LAL causes accumulation of fats leading to liver damage, feeding issues in infants and mortality

hydrolysis of fatty acid esters

Degrades neutral lipids at an acidic pH in lysosomes. Crucial for cell membranes and storing energy.

In frame mutation 894G>A occurs at an exon splice junction resulting in on 3-5% of normally spiced enzyme product.

enzyme replacement therapies

Recombinant lysosomal acid lipase (sebelipase alfa)

Recombinant LAL protein produced in hens eggs white can now be used successfully as treatment

advantages to enzymes

• High quality, specific reactions with few side products

• Capacity to accelerate chemical processes and increase yields

• Often straightforward separation of catalysts from products and high purity

• Water based green chemistry - enzymes reduce toxic waste from conventional chemistry and are themselves biodegradable

• Lower reaction temperatures - lower energy consumption

• Often catalyse stereoselective reactions