Protease & Enzyme Activity

FRET substrates

FRET occurs between a peptide tagged to a donor and an acceptor when placed within 10-100Å of each other
resulting in the donor’s excitation fluorescence to be quenched by the acceptor.
Enzymatic hydrolysis of the peptide results in recovery of the donor fluorescence following spatial separation of the donor and acceptor upon energy transfer.

Fluorogenic substrates

These substrates do not require a quencher and contain a C-terminal dye that does not fluoresce
until it is cleaved from the peptide (fluorescent form of dye is released).

Why is Protease Activity Important?

Protease enzymes catalyze the breakdown of proteins into smaller peptide fragments and/or single amino acids.
Endopeptidases are proteases that cleave internally of the protein sequence,
and those that cleave at the N-terminus or C-terminus are exopeptidases.

Endopeptidases are categorized into 4 classes according to the catalyst type (amino acid or metal)
that is located within the active site that drives the hydrolytic breakdown of the substrate peptide bond, including
(a)Serine Proteases (Trypsin, Elastin, Thrombin, HCV NS3/4A), (b)Cysteine proteases (Caspase, Cathepsin),
(c)Aspartyl Proteases (HIV, Renin), and (d) Metalloproteases (MMPs).

Through very specific and controlled protein substrate cleavage, proteases help in the initiation, modulation and termination
of many crucial cellular functions, including DNA replication, cell proliferation, wound healing, immunity, angiogenesis and apoptosis.
Proteases are involved in many diseases caused by aberrant metabolic protease activity or
through infection by pathogens that express proteases for their infectivity or replication.