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Proteases are essential for any living system and plays an important role not only in the degradation of proteins but also in various biological processes such as blood clotting, digestion, apoptosis etc., Proteases occur in a wide range of organisms ranging from fungi to humans. Though proteases are best characterised in the human genome, the exact mechanism of their function still remain to be fully understood. With time many mechanisms for their actions were proposed but these mechanisms are still being debated and new mechanisms are being put forth. This review aims at discussing the various types of proteases and elucidating their mechanisms.
Proteases are proteolytic enzymes that function as molecular knives and cut long protein sequences into shorter fragments. Proteases are highly selective in the sense that a particular protease cleaves a certain amino acid sequence and they do not cleave randomly. Approximately 2% of human genome codes for proteases. Proteases perform a variety of biological processes such as ectodomain shedding of cell surface proteins, regulating the activation/inactivation of important biomolecules like cytokines, hormones, growth factors, replication, transcription, regulation of protein synthesis, and cell cycle regulation. Besides this, proteases are also important in regulation of birth, conception, digestion, ageing etc. Thus due to their important roles in various biological processes, proteases have become great interests for medical research and pharmaceutical industry for drug development. (Laskar and Chatterjee. 2009)
History of proteases goes back down as early as the 1930s when Bergmann investigated the specificity of proteases and the reversal of proteolysis. At that time proteases were considered an impurity and were removed during protein isolation. But in the year 1938 Northrop et al demonstrated that proteases were chemically pure and obeyed the thermodynamic criteria for pure compounds. However many scientists were not able to investigate their role as proteases were not available commercially and due to technological backwardness it was not easy to isolate them and study. But with the advent of new technologies, the research in this field spread like a wildfire and by the 1970s the mechanism of many proteases such as chymotrypsin, pepsin, carboxypeptidase were identified.(Neurath 1999)
Proteases can be classified by their site of action and by their reaction mechanism as follows:
By site of action they are classified into
By reaction mechanisms and nature of active site residues involved in mechanism
they are classified into
Glutamic acid Proteases
They are named serine proteases because all the proteases belonging to this class involve a serine in catalysis. The mechanisms of these proteases involve a charge relay system and catalytic triad of amino acids such as Serine, Aspartic Acid and Histidine. Different members of this family make use of the same catalytic triad in different positions. For eg Bacillus licheniformis subtilisin uses Ser221, His64 and Asp32. Some unconventional serine proteases also use of a totally different catalytic triad. For eg., Ser/Glu/Asp in Sedolisin protease (Wlodawer, et al. 2001)
Serine acts as a nucleophile and attacks the peptide bond of the substrate to form a tetrahedral intermediuate. Imidazole group acts as an acid first to protonate leaving nitrogen. Aspartate stabilizes the positive charge on Histidine. Histidine then acts as a base to abstract the proton from water and make it a strong nucleophile. Water catalyses the hydrolysis of acyl enzyme and leads to the product formation.
Mechanism Of Chymotrypsin
The substrate binds to the free enzyme and the attack on the peptide bond is facilitated as the side chain residue adjacent to the peptide bond docks at the hydrophobic site. OH of serine acts as a nucleophile and attacks the carbonyl carbon of the substrate leading to the formation of tetrahedral intermediate (2). The carbonyl oxygen becomes negatively charged in this process and is stabilized by hydrogen bonding with NH group of Ser195 in the oxyanion hole. However this stability is short lived and instability leads to the collapse of tetrahedral intermediate. The carbon-oxygen double bond is reformed and in this process the carbon-nitrogen bond in the peptide is broken. This nitrogen is protonated by His57. This leads to the formation of acyl enzyme complex (3). Now His57 acts as a base and abstracts a proton from water thus making water a strong nucleophile. This now attacks the ester linkage of the acyl enzyme intermediate and carbonyl oxygen once again develops a negative charge and a second tetrahedral intermediate is formed(4,5). Again instability of the charge leads to the collapse of the intermediate thereby displacing Ser195 (6). The product then diffuses from the active site regenerating the free enzyme (7). (See Diagram Below) Chymotrypsin cleaves aromatic amino acids such as Phe, Tyr, Trp from the C-terminal end.(Lehninger, Nelson and Cox 2005)
Various Hypothesis On The Mechanism Of Serine Proteases
Although the charge relay mechanism is widely accepted to be involved in the working of serine proteases, the issue whether there is one proton transfer or two proton transfers is highly controversial. It has been highly debated whether the proton in the Asp102-His57 hydrogen bond resides on His57 or on Asp102. In the year 1967, Matthews and his colleagues (Matthews, et al. 1967) explained that the catalytic site of chymotrypsin has an aspartic acid bound to histidine which is in turn bound to serine. The negative aspartic acid residue polarizes this system and this made oxygen of serine highly nucleophilic and reactive towards amides and esters. This explanation was later refined and it was suggested that two protons are transferred i.e. one from Ser 195 to His 57and the next from His 57 to Asp 102 resulting in the formation of a tetrahedral intermediate and neutral Asp 102 and His 57. This mechanism was also highly contentious as it requires the pKa of Histidine to be lower than Asp102 or in other words the pKa of His 57 must decrease during the formation of tetrahedral intermediate which is quite contrary to NMR studies and Nuclear diffraction experiments which demonstrate that the pKa of Histidine 57 incerases to 10 during the formation of tetrahedral intermediate. Therefore the scientists strongly advocate that only one proton transfer exists and protonated His57 is in an electrostatic bonding with Asp102. (Hedstrom 2002)
Another hypothesis was the ring flip hypothesis. It was widely accepted that during serine protease catalysis, His 57 abstracts a proton from Ser195 and donates it to the leaving amine group and thereby acting both as a base and acid. But scientists argue that if this is the case then His57 H+ should be close to Ser195 in the tetrahedral intermediate and could donate the proton back to Serine resulting in the regeneration of the substrate. Therefore for the reaction to proceed forward, His57 must be positioned near the leaving amine group. It achieves this by doing a flip thereby placing the ND1 proton near the leaving group after the formation of tetrahedral intermediate as shown in the diagram. This is the His Flip hypothesis. (Hedstrom 2002)
Aspartic proteases belong to the pepsin family and are active at acidic pH (1.9-4). They involve aspartic acid in their catalysis and hence the name. They are found in mammals, plants, fungi and retroviruses. They carry out a variety of functions such as protein digestion, blood pressure regulation, and viral protein synthesis for the survival of certain viruses.
Early research investigating the mechanism of Aspartic Acid proteases implicated that they functioned via an amino enzyme intermediate involving a covalent peptide bond. However scientists were not able to trap the covalent intermediate. Moreover NMR and X-Ray crystallographic studies ruled out the possibility of a covalent intermediate due to the steric hindrance posed by it. In the year 1985 James et al deduced a mechanism without the involvement of a covalent intermediate. However this mechanism was highly contended as it involved simultaneous protanation of both carboxyl groups which was not feasible as pH greater than 2 would not be consistent with the pKa of the carboxyl groups which are less than 2 and between 4 and 5 respectively. (James and Sielecki 1985)
Thus a new catalytic mechanism was proposed. In this mechanism, similar to the catalytic triad, Asp32 and Asp215 constitute a functional unit. In the free enzyme the diad proton is bound to either amino acid as the carboxyl groups are symmetrical. When the substrate binds the diad carboxylate accepts the the proton of water, thus acting as a base. Also the diad proton is donated to the carbonyl oxygen of the substrate leading to a development of a negative charge on Asp215. In the next step the protonated carbonyl oxygen donates the proton to the carboxyl group of Asp32 and the diad proton protonates the leaving amine and also breaks the C-N bond, resulting in the formation of products. Therefore in this mechanism the leaving nitrogen is protonated by the proton from water (nucleophile) via the diad and not by the protonated oxyanion. Thus this mechanism depicts the push-pull acid base catalysis. The general mechanism is illustrated below. (Polgár 1987)
Mechanism Of HIV Protease
The mechanism of HIV proteases involves two aspartate residues Asp25 and Asp25'. In this mechanism Asp25' is in unprotonated form and Asp25 forms a proton hydrogen bond with the carbonyl oxygen of the substrate. Asp25' acts a general base and accepts a proton from water thus converting water to a strong nucleophile. Water now attacks the carbonyl carbon of the peptide group. This results in the formation of amine dihydrate which is the transition state and in this stage there are two proton transfers. One involves the transfer of proton from Asp25' to the leaving amine group of proline and the other aspartic acid acts as a base and accepts a proton from one of the hydroxyl groups. These proton transfers leads to collapse of the transition state and generates the products and the free enzyme. The enzyme now has reversed in protonation i.e. Asp25 becomes unprotonated and Asp25' becomes protonated.(Garrett and Grisham 2005)
Various Hypothesis On Aspartate Proteases
In the year 2001 Northrop et al proposed that the formation of a low barrier hydrogen bond between Asp residues plays an important role in the catalytic function. The mechanism proposed was similar to the previous mechanism but for the addition of an isomerisation step to restore the cyclic structure and to reform the low barrier hydrogen bond. In the first step of this mechanism the substrate forms a lose complex with the enzyme. Enzyme then closes the flap around the substrate so that the substrate becomes properly oriented. The bound water then loses its proton and becomes a nucleophile which attacks the carbonyl carbon of the substrate. One of the Asp residues then donates a proton to the leaving nitrogen. This is followed by bond cleavage and opening of the flap to release the products. The other Asp is then deprotonated and water molecule reattaches and the low barrier hydrogen bond is reformed. (Dunn 2002)
A further modification of the catalytic mechanism was proposed by Andreeva et al. They proposed the probability of another water molecule in the active site and they designated it as W2. W1 is the water which is already said to exist in the active site. W2 lies between Tyr75, Asn37 and Ser35 and is usually bound to carbonyl oxygen of Asn37 and OH of Ser35 in the free state. There is a Thr218-Asp215 hydrogen bond which helps to maintain the negative charge on Asp215.
When the substrate approaches, Thr218-Asp215 bond breaks. Asp215 now acts as a base accepting a proton from water W1 and facilitating the nucleophilic attack of water on carbonyl oxygen of the substrate leading to the formation of tetrahedral state. Asp32 acts as an acid and donates a proton to the carbonyl carbon of the substrate and Ser 35 forms a new hydrogen bond with Asp32. W2 then donates a hydrogen bond to oxygen of Ser35 and itself accepts a bond with Tyr75. A new hydrogen bond is formed between Trp39 and side chain oxygen of Tyr75. This helps to donate a hydrogen bond to W2. The orientation of W2 changes due to binding between the NH group of Trp39 and OH group of Tyr75. The final step of this mechanism is similar to other postulated mechanisms in which Asp215 protonates the leaving nitrogen and Asp32 accepts a proton to form the products and the free enzyme is restored. (Dunn 2002)
Cysteine proteases are another type of proteases. These proteases have a cysteine and histidine in their active sites. They are found in mammals, plants, fungi, viruses, bacteria and protozoa. They carry out a variety of functions such as extra cellular matrix remodelling, intracellular thyroglobin processing for releasing thyroid hormones, apoptosis, defribrinating wounds etc.,
Mechanism of Cysteine Proteases is similar to that of serine proteases except that the thiol group of cysteine attacks the nucleophile instead of serine. This is shown in the diagram below.
Mechanism Of Papain-a cysteine protease
The mechanism involves the role of Cys25 and His 159 in the active site though there are serious doubts being raised whether these two amino acids are sufficient for the catalytic activity. In the first step the imidazole H+ group of the histidine polarizes the SH group of Cysteine to form a highly nucleophilic thiolate-imidazolium ion pair (a). This thiol anion attacks the carbonyl carbon of the substrate (b) leading to a formation of Tetrahedral intermediate (c). Similar to chymotrypsin the oxyanion formed is fixed to the oxyanion hole. The oxyanion is stabilized by hydrogen bonding to NH of Cys25 and amino group of Gln19. This esterification also causes the imidazole group as a general acid and it donates its protons to the leaving nitrogen group leading to the production of an acyl enzyme (d). It is important to note that protonation does not occur as such but happens only when His159 is rotated. During deacylation, the imidazole nitrogen acts as a base and abstracts a proton from water thereby facilitating the nucleophilic attack of water on the carbonyl carbon of the acyl enzyme (e). A second tetrahedral intermediate is formed and this collapses due to charge instability and produces the regenerated enzyme and the products (f, g). Papain generally cleaves Lysine and Arginine from the C terminal end. (Otto and Schirmeister 1997)
The exact mechanism of cysteine proteases is still being evolved. There was initially a school of thought that a third amino acid such as Asp158 was also involved in the catalytic mechanism. However this theory was disproved as Asp158 was found to be located far from the active site and also that Asp158 was missing in many cysteine proteases. Scientists have also proposed the role of Asn175 in directing the imidazole to various optimum positions during hydrolysis and stabilization of ion par by positioning the imidazole appropriately. But these studies are still being investigated and a clear picture of cysteine protease mechanism is not available. (Otto and Schirmeister 1997)
Metalloproteases are enzymes which have metal ions such as Zn, Co in their active site. They are found mostly in mammals, plants, fungi and retroviruses. They carry out a variety of functions such as post translation modification, blood clotting, wound healing and reproduction, tissue remodelling and repair, tumour metastasis etc,
In these proteases zinc bound water acts as a nucleophile which attacks the carbonyl bond to form a pentacoordinated intermediate. Glutamic acid then acts as an acid and donates a proton to leaving nitrogen leading to the formation of products.
Mechanism Of Thermolysin
In the free enzyme zinc has a coordination number of 4. The ligands are from His 142, His 146, Glu 166 and water. When the substrate approaches the zinc bound water forms a hydrogen bond with Glu 14 and the oxygen is liganded to Zn2+. This is followed by the formation of Michaelis complex where the carbonyl oxygen of the substrate hydrogen bonds with His231 and zinc bound water. This type of interaction facilitates the nucleophilic attack of water on the carbonyl carbon resulting in the formation of a pentacoordinate intermediate (b). During this process a peptide hydrate is formed and one of the oxygen in this hydrate is stabilized by hydrogen bonding to both His231 and Tyr157. The second oxygen by hydrogen binding with Glu 143 and interacting with Zn stabilizes itself. Asn112 and Ala113 form hydrogen bonds with the nitrogen to form a tetrahedral intermediate(c). In the next step Glu 143 acts as a general acid and donates a proton to the leaving nitrogen and causes the tetrahedral intermediate to collapse. This results in the formation of products (d). (Matthews 1988)
The sixth type of proteases belonging to the G1 family in MEROPS database are the Glutamic proteases. They are found prominently in fungi. They are also known as Eqolisins since they have glutamic acid and glutamine in their active sites. (Laskar and Chatterjee, 2009)
Mechanism As Exemplified By Scytalidocarboxyl Peptidase B
Mechanism is exemplified from the enzyme scytalidocarboxyl peptidase B (SCP-B). This consists of 2 amino acids namely Glu136 and Gln53. Water is bound between these amino acids. Initially the enzyme-substrate complex would have conformation angles of Φ ~ -112° and Ψ~ - 90°. A nucleophilic attack of water is facilitated by the assistance of Glu 136 which acts as a general base. Due to this attack the carbonyl carbon becomes tetrahedral and the Ψ angle decreases to ~ - 90° thereby becoming fully staggered in the intermediate. The side chain of Gln53 NÆ2 interacts with the carbonyl oxygen of the substrate via a hydrogen bond thereby polarizing the carbonyl bond of the peptide. The interaction of Gln53 helps in the formation of the tetrahedrahedral intermediate and in the stabilization of the oxyanion formed in the intermediate. Glu136 then acts as a general acid and donates a proton to the leaving group nitrogen leading to the collapse of tetrahedral intermediate and in the formation of products. (Fujinaga, et al. 2004)
These proteases have a Thr in their active site. This group of proteases is well exemplified by Proteosomes. They are found in bacteria, fungi, yeast and mammals. They play an important role in the ubiquitination and proteosomal degradation of proteins.
Mechanism Of Proteosome
Firstly there is an autocatalytic maturation of active sites. The substrate first comes and positions itself in the active site via Hydrogen bond. Lys33 helps in lowering the pKa of the amino group of Thr1 and helps Thr1 to act as a general base and accept a proton. The nitrogen of Thr1 accepts a proton from water and Thr1-Ogamma binds to the carbonyl oxygen of the substrate. This leads to the formation of a tetrahedral intermediate. In the tetrahedral intermediate proton from Nitrogen atom of Thr1 is transferred to áµž oxygen atom with the help of water. This leads to the formation of acyl enzyme intermediate. Water now acts as a nucleophile and attacks the carbonyl oxygen of the substrate attached to Thr1 áµž oxygen atom. This releases the amino component of the substrate. The acyl-enzyme undergoes hydrolysis to give the acidic component of the substrate and regenerating the active site. (Marques, et al. 2009)
From being considered an impurity to an integral part of the biological system, proteases have come a long way. With the advent of X Ray Crystallography, NMR and site directed mutagenesis scientists are trying to figure out the mechanism of action of proteases. Based on the current understanding of the mechanism of proteases, inhibitors are now being developed to fight various types of fungal, viral and parasitic infections. A number of proteases associated with toxins such as Clostridium tetani and Bacillus anthracis are also being investigated for developing potential vaccines against these toxins. Human proteases are now being used to predict the prognosis of cancer. For eg., kallikreins as an indicator in prostate cancer.
Though there have been remarkable breakthroughs in identifying the amino acids involved in the catalytic mechanism and detecting the transition states in the past four decades, scientists have not yet come up with an exact mechanism of action for proteases. Many mechanisms, no doubt have come up, but are highly contended. Scientists are continuing to explore the world of proteases for a better understanding of their working mechanism.