Papain

General Structural Features

Papain is a relatively simple enzyme, consisting of a single 212 residue chain. A majority of papain's residues are as shown in purple. As with all proteins, it is primarily the exclusion of these residues by water that leads to papain's assumption of a globular form. Despite its apparent simplicity and small size, papain folds into two distinct, evenly sized , each with its own (surface residues are transparent, hydrophobic-core residues are colored and opaque, and the remaining are polar, non-surface residues).^[7] These two subunits are held together with , where each protein domain holds the opposite domain. In papain's case the "arm" crossing primarily occurs on or near the surface.^[8] It is between these two domains that the is situated. The two domains interact with one another via hydrophobic interactions, (shown in white), and electrostatic interactions in this cleft. For example, from the L Domain hydrophobically interacts with the carbon atoms on residues Lys-174, Ala-162 and Pro-129 of the R Domain. hydrogen bonds multiple times with the oxygen atoms of Ser-176 and also with the oxygen atom on Tyr88. Electrostatic interactions are seen between where the carboxyl group of Glu-35 forms an ionic bond with the ammonia group of the Lys-174 residue. The of interactions within the cleft between the two domains ensures that the lobes do not move with respect to one another. ^[9]

In addition to hydrophobic residues, papain contains a variety of , some carrying a , shown in gray at physiological pH, and are therefore acidic; others a , shown in purple, and are therefore basic. The rest of the , shown in a light gray, are neutral. As expected, the charged face outward due to their hydrophilic nature.

Papain's secondary structure is composed of 21% (45 residues comprising 17 sheets) and 25% (51 residues comprising 7 helices). The rest of the residues, accounting for over 50% of the enzymes structure, make up ordered non-repetative sequences.^[10] These secondary structures may be traced from the N- to C-terminus by means of . As shown in this scene, the red end begins the protein at the N-terminus, and can be traced through the colors of the rainbow to the blue end at the C-terminus. These secondary structures form as a result of favorable hydrogen bonding interactions within the polypeptide backbone. Meanwhile, secondary structures are kept in place by hydrophobic interactions and hydrogen bonds between sidechains of adjacent structures. For example, the (residues 25-42) is maintained as a result of between backbone carbonyl atoms and the hydrogen on the amide nitrogen four residues away. However, are present between this helix and the rest of the protein, suggesting that this helix is coordinated primarily by hydrophobic interactions. This is reasonable given its central location in the enzyme. As expected, the helix contains many (red residues are hydrophilic).

strongly contribute to the tertiary structure of papain.^[5] In this particular image, clarification of residue coordination is demonstrated by color: paired residues are shown in the same color, oxygen is shown in red, and nitrogen is shown in blue. The tertiary structure of papain is also maintained by three , which connect , , and ^[4]. These disulfide bonds are likely important in conserving the structural integrity of the enzyme as it operates in extracellular environments at high temperatures.

Substrate Binding

Papain has a relatively small consisting of three residues: Cys-25, His-159, and Asn-175. The sulfhydryl group on Cys-25 often forms covalent bonds with substrates. His-159 supports Cys-25, and while Arg-175 does not directly participate in the catalytic mechanism, it keeps histidine-159 in its stabilized imidazole form. In addition to the active site, sometimes referred to as the catalytic site, Papain consists of many Subsites are defined as the regions on the enzyme surface which interact with one amino acid residue of the substrate. In the case of Papain, there are seven subsites labeled as follows: S1, S2, S3, S4, located on the amino side of the catalytic site; S1', S2', and S3', located on the carboxyl side of the catalytic site. The binding sites of a substrate are labeled according to how they fit into the binding cleft. P1 associates with S1, P2 with S2, etc. All seven of Papain's subsites hydrogen bond to the corresponding substrate P subsites. The following of Papain have been identified as follows: S1 - His-159; S2 - Trp-177; S3 - Gln-19; S4 - Gly-23; S2' - Asp-158; and S3' - Asp-64. Unfortunately, S1' has yet to be identified.

The seven subsites of Papain have various preferences for substrate residues. Through a variety of experiments, Berger & Schechter^[11], were able to show that S1 binds alanine better than glycine, and the larger side chains of lysine, arginine, leucine, and phenylalanine better than alanine. Thus showing that binding in S1 is predominantly hydrophobic. S2 prefers a phenylalanine or a valine residue. Interestingly enough, S2 binds to hydrophobic residues of both short and long peptide chains. They were able to show that subsites S1' and S2' are strongly stereospecific. The conclusion of their research was that Papain's binding site residues show a strong stereospecificity, special interactions, and space limitations.^[12]

Catalytic Mechanism

It was once thought that cysteine proteases, like serine proteases, contained a , consisting of Cys-25, His-159, and Arg-175 ^[13]. However, site-directed mutagenesis-based studies have demonstrated that Arg-175 is not directly involved in catalysis. Although Arg-175 is clearly important for the enzyme's activity (an Arg175Ala mutation reduces its activity to undetectable levels), this residue neither reacts with the substrate nor modulates the pKa of reacting residues, and therefore cannot be considered catalytic.^[14][15] Arg-175 is believed to keep histidine-159 in its stabilized imidazole form, while both histidine-159 and cysteine-25 take part in the actual catalytic mechanism.^[16] Despite this, the basic mechanism of papain-catalyzed proteolysis proceeds much like that of serine proteases. The mechanism begins when a peptide binds to the active site. Cys-25 is then deprotonated by His-159 and attacks the substrate carbonyl carbon. This forms a covalent, tetrahedral intermediate that is stabilized by an , formed in large part by Gln-19. Next, His-159 acts as a general acid, protonating the nitrogen in the peptide bond, which acts as a leaving group as the carbonyl reforms. This now free C-terminal portion of the peptide is released. Water then enters the active site and attacks the carbonyl carbon while it is deprotonated by His-159, again forming an oxyanion hole-stabilized tetradral covalent intermediate. Finally, the carbonyl reforms and the Cys-25 sulfur acts as a leaving group, releasing the N-terminal portion of the peptide and regenerating the enzyme. This entire mechanism is shown below^[17].:

General mechanism of papain catalysis ^[16]. Arg-175, which orients His 159, and Gln-19, which contributes to the formation of the oxyanion hole, are not shown.

Leupeptin

is a commonly studied broad-spectrum competitive protease inhibitor first crystallized by Schröder et. al. It inhibits by binding and interacting with the active site which allows it to block the enzyme's desired protein substrate from binding. There are many that interact with leupeptin, which predominantly interact (interactions in blue), including tyrosine, tryptophan, and valine, which coordinate the bound leuptin. Some of the enzyme's residues also make . These hydrogen bonds, shown in white, include interactions between hydrogens on both Gln-19 and the amide nitrogen of the catalytic Cys-25 with the arginal carbanion, forming the catalytically important oxyanion hole. In addition, Gly-66 interacts with the second leucine in leupeptin while Asp-158 interacts with a hydrogen on the arginal. These interactions further stabilize and orient the substrate in the binding pocket^[18].

A recent study has shown that Leupeptin forms a covalent bond between its and the hydrogen in Cys-25. The inhibitor has the structure Ac-Leu-Leu-Arginal, where Ac is an acetyl group attached to the nitrogen of the first leucine. Cysteine-25, which acts as a catalytic nucleophile, attacks the arginal aldehyde forming a tight-binding transition state from which the normal catalytic mechanism cannot proceed due to this carbonyl having no potential leaving groups bonded to it.

Cathepsin K

The goal of research for the development of an inhibitor for is the hope to develop a treatment for osteoporosis. In two different Cathepsin K inhibitors, , 1BP4, and , 1BQI. 1BP4, N-[(benzyloxy)carbonyl]-L-leucyl-N-[(2S)-1-hydroxy-4-methylpentan-2-yl]-L-leucinamide, inhibits by interacting with on papain: Gln-19, Gly-20, Ser-21, Gly-23, Asn-64, Gly-65, Gln-142, Asp-158, His-159, Trp-177, and Trp-181. These interactions range from hydrophobic, electrostatic, and hydrogen bonding, to , illustrated in blue, between the aromatic ring of the carbobenzyl group on 1BP4, and Trp-177 of papain. The inhibition of papain by IBQI, carbobenzyloxy-(L)-leucinyl-(L)leucinyl methoxymethylketone, is quite similar to that of IBP4, although it does not bind quite as tightly. It binds to of papain: Gln-19, Gly-23, Gly-65, Gln-142, His-159, Trp-177, Trp-181. Additionally, it has similar , shown in blue, between the Cbz ring on the inhibitor and Trp-177.^[19]

Cathepsin L

, another inhibitor of Papain, is an endosomal cysteine protease that is believed to have both physiological and pathophysiological effects on the human body. It has been indicated not only in cancer, rhematoid and osteo arthritis, and Alzheimer's, but its mechanism also appears similar to that of Ebola, SARS, Leishmania, malaria, Chagas' disease, African trypanosomiasis, toxoplasmosis, and amoebiasis.^[20] Understanding the mechanism of inhibition through the use of papain is therefore crucial to developing treatments for such diseases.^[21] Cathepsin L interacts with the Gln-19, Cys-25, Gly-66, Asp-158, and Trp-177 by hydrogen bonding them (Cathepsin L is illustrated in CPK coloring while the interacting sites of Papain are also shown in CPK). In addition to hydrogen bonding, hydrophobic interactions exist to exclude water, allowing the papain enzyme and Cathepsin L to associate even closer.

An interesting inhibitor for Cathepsin L developed using papain as the model protease is that of .^[22] It forms a with Cys-25. Five other , labeled in white, are also involved in the bonding of Clik-148 to papain: Gln-19, Gly-66, Asp-158, Trp-177, and Ser-205. These participate in hydrophobic, , and that effectively fill up the cleft between the two domains of papain.^[23]

Stefin B

acts as a competitive inhibitor to cysteine proteases - it binds tightly but reversibly to the Papain active site. Its interaction is much more complicated than many other cysteine protease inhibitors, such as those illustrated above. Stefin inhibitors are characterized by an M_r of about 11,000, with no disulfide bonds and no associated carbohydrates.

In Stefin B, the Gly-9 residue along with , illustrated in magenta, form a "wedge" complementary to the active site groove of Papain. This wedge makes extensive and tight interactions with Papain which involves the embedding of 16% of Stefin B into Papain with a total of 128 intermolecular atom-atom interactions occuring. Met-6 - Pro-11, Gln-53 - Asn-59, Gln-101 - His-104, Tyr-124 and Phe-125 on the wedge all have some interaction with the enzyme, though Cys-25 is the only one to form a direct contact. All residues from the base and both sides of the are involved in the complex with the inhibitor.

There are a small number of between Stefin B and Papain, however there are many more polar interactions mediated by . Thirteen solvent molecules bridge polar residues of the enzyme and inhibitor. Seventeen hydrogen bonds are made with a solvent molecule and Stefin B. Fourteen of these bridges form a Papain contact. The rest of the interactions are largely hydrophobic-- involving apolar .^[24]