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B4. Proteases Mechanisms

Serine proteases are just one type of endoproteases. However, they are extremely abundant in both prokaryotes and eukaryotes. Protease A, a chymotrypin-like protease from Stremptomyces griseus, has a very different primary sequence than chymotrypsin, but its overall tertiary structure is quite similar to chymotrypsin, The positions of the catalytic triad amino acids in the primary sequences of the protein are very similar, indicating that the genes for the proteins diverged from a common precursor gene. In contrast, subtilisin, a serine protease from B. Subtilis, has both limited sequence and tertiary structure homology to chymotrypsin. However, when folded it also has a catalytic triad (Ser 221 - His 64 - Asp 32) similar to that of chymotrypsin (Ser 195 - His 57 - Asp 102). 

Jmol: Updated Comparison of Chymotrypin and Subtilisin Jmol14 (Java) | JSMol (HTML5) 

Proteases have multiple functions, other than in digestion, including degrading old or misfolded proteins and activating precursor proteins (such as clotting proteases and proteases involved in programmed cell death). In general, four different classes of proteases have been found, based on residues found in their active sites. Proteases can also be integral membrane proteins, and carry out their activities in the hydrophobic environment of the membrane. For example, aberrant cleavage of the amyloid precursor protein by the membrane protease presenillin can lead to the development of Alzheimers.

Figure: amyloid precursor protein


Classification of Proteases
Class (active site) Active Site Nucleophile Location Examples
Serine/Threonine Hydrolases Ser/Thr soluble trypsin, chymotrypsin, subtilisin, elastase, clotting enzymes, proteasome
membrane Rhomboid family
Aspartic Hydrolases H2O activated by 2 Asps soluble pepsin, cathepsin, renin, HIV protease
membrane β-secretase (BACE), presenilin I, signal peptide peptidase
Cysteinyl Hydrolases Cys soluble bromelain, papain, cathespsins, caspases
membrane ?
Metallo Hydrolases H2O activated by 1 or 2 metal ions soluble thermolysin, angiotensin converting enzyme
membrane S2P family
Glutamate Hydrolases Glu . eqolysins (fungal)
Asparagine Lysases (EC4) (elimination rx which are self-cleavage and hence not catalytic) Asn . Tsh autotransporter E. Coli

β-secretase (BACE) is a membrane protein that contains two necessary Asp residues in its ectodomain (extracellular domain) which are used in the first cleavage of the N terminal domain of the beta amyloid precursor protein to release a soluble, N-terminal fragment of about 100,000 MW. γ-secretase, necessary for the second cleavage which frees the Aβ peptide is a heterotetramer composed of presenillin-1, nicastrin, APH-1 and PEN-2, and is located in neural plasma membranes and endoplasmic reticulum. The Aβ peptide moves to the extracellular side of the neural membrane where it aggregates. The remaining cytoplasmic part of the beta-amyloid precursor protein may regulate transcription. The presenillin subunit has protease activity. γ-secretase also cleaves another cell surface receptor protein, Notch. When this receptor has bound an extracelluar ligand, γ-secretase cleaves Notch within the cytoplasm, and the released fragment modifies gene transcription. The APH-1 subunit appears to inhibit presenilin protease activity while PEN-2 promotes it. Inhibiting γ-secretase would be an effect treatment for Alzheimers, but might have serious side effects since Notch processing would also be affected.

Figure: Cleavage of beta amyloid precursor protein: protease and cofactors


The γ-secretase protein quartet, and its roles in brain development and Alzheimer's disease. Presenilin-1, nicastrin, APH-1 and PEN-2 form a functional γ-secretase complex, located in the plasma membrane and endoplasmic reticulum (ER) of neurons. The complex cleaves Notch (left) to generate a fragment (NICD) that moves to the nucleus and regulates the expression of genes involved in brain development and adult neuronal plasticity. The complex also helps in generating the amyloid β-peptide (Aβ; centre). This involves an initial cleavage of the amyloid precursor protein (APP) by an enzyme called BACE (or β-secretase). The γ-secretase then liberates Aβ, as well as an APP cytoplasmic fragment, which may move to the nucleus and regulate gene expression. Mutations in presenilin-1 that cause early-onset Alzheimer's disease enhance γ-secretase activity and Aβ production, and also perturb the ER calcium balance. Consequent neuronal degeneration may result from membrane-associated oxidative stress, induced by aggregating forms of Aβ (which create Aβ plaques), and by the perturbed calcium balance. Reprinted by permission from Nature: Mattson, M. Nature. 422, 385-387 (2003)

How do integral membrane protease catalyze the hydrolysis (using water) of transmembrane domains in proteins, given the hydrophobic environment of the bilayer? The rhomboid class of membrane proteases, which are found in prokaryotic and eukaryotic cells, is one of the most conserved membrane proteins in nature. Sequence comparison of rhomboid proteins shows a possible catalytic serine residue that is buried to the same depth in the bilayer as the cleavage site for rhomboid protein substrates. In addition the proteases share conserved amino acids similar to the catalytic triad of soluble serine proteases, with the critical amino acid localized on different membrane-spanning regions of the protein. The membrane triad amino acids are Ser, His and Asn (instead of Asp as in classical serine proteases). Classical serine protease inhibitors prevent cleavage of membrane protein substrates, but given the difficulty of reconstituting a functional purified rhomboid protein in vitro, the identity of specific amino acids in the catalytic mechanism is inferential at present. 

Helical wheel diagrams of putative transmembrane domains of the rhomboids (determined through hydropathy plots) show several to be amphiphilic. This might allow water, necessary for hydrolysis, to enter into the catalytic active site of the enzyme, although as was noted before, water has a reasonably high permeability through a membrane bilayer. The chief requirement for protein substrates of rhomboids is the presence of a transmembrane domain in the target protein. No specific amino acid sequence seems to be required for specificity of one particular substrate, the drosophila transmembrane protein spitz found in Golgi membranes. On cleavage of this protein, the remaining part of the protein is released as a water soluble protein to the lumen of the Golgi where it can eventually be released from the cell. The soluble protein fragment that is released from the cell contains an epidermal growth factor domain. 

Recently the structure of a rhomboid protease, GlpG, from E. Coli, was determined. This transmembrane protein has 6 transmembrane helices. The enzyme has a polar active site at the bottom of a V-shape opening situated laterally in the membrane. Active site His and Asn residue and many water molecules are deep in this V-shaped cleft well below the surface of the membrane. Access to the transmembrane strand of the protein substrate is blocked by a loop, which must be gated open to allow substrate access between the V-shaped gap between helices S1 and S3. Ser 201 (nucleophile) and His 254 (general base/acid) are essential for activity. 

Jmol: Updated E. Coli GlpG rhomboid intramembrane protease Jmol14 (Java) | JSMol (HTML5)