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 Lys174, Ala162 and Pro129 of the R Domain. hydrogen bonds multiple times with the oxygen atoms of Ser176 and also with the oxygen atom on Tyr88. Electrostatic interactions are seen between where the carboxyl group of Glu35 forms an ionic bond with the ammonia group of the Lys174 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.

Active Site and Substrate Binding

^[11]

Catalytic Mechanism

It was once thought that cysteine proteases, like serine proteases, contained a , consisting of Cys-25, His-159, and Arg-175 ^[12]. 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.^[13][14] 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.^[15] 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 oxyanion hole, formed in large part by . 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^[16].:

General mechanism of papain catalysis ^[15]. Arg-175, which orients His 159, and Gln-19, which contributes to the formation of the oxyanion hole, are not shown.

Leupeptin

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 active site residues</scene> that interact with Leupeptin and they predominantly interact . In addition to hydrophobic interactions, there are also hydrogen bonds holding Leupeptin in the active site. For example, the residues Gln 19 and CYS 25 form with the Leupeptin molecule.

A recent study has shown that Leupeptin forms a covalent bond between its and the sulfur 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. The catalytic nucleophile (cysteine in papain) 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. Analysis of the resulting structure revealed that the of papain consists primarily of a variety of , including tyrosine, tryptophan, and valine, which coordinate the bound leuptin. Some of the enzyme's residues also make with some of the leupeptin atoms. These hydrogen bonds, shown in yellow, include interactions between both 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 interaction further stabilize and orient the substrate in the binding pocket^[17].

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, referenced PDB codes and , it is evident that the inhibitor binds with much closer proximity than that of Clik148. 1BP4 is a cathepsin K inhibitor, N-[(benzyloxy)carbonyl]-L-leucyl-N-[(2S)-1-hydroxy-4-methylpentan-2-yl]-L-leucinamide, that inhibits by interacting with 11 different residues on papain: Gln19, Gly20, Ser21, Gly23, Asn64, Gly65, Gln142, Asp158, His159, Trp177, and Trp181. These interactions range from hydrophobic, electrostatic, and hydrogen bonding, to between the aromatic ring of the carbobenzyl group on 1BP4, and TRP177 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 seven residues of papain: Gln19, Gly23, Gly65, Gln142, His159, Trp177, Trp181. Additionally, it has similar between the Cbz ring on the inhibitor and Trp 177, though it is more difficult to visualize with the given PDB file.^[18]

Cathepsin L (PDB ID #: 1CVZ)

Cathepsin L is another inhibitor of the papain enzyme. Cathepsin L interacts with the Gln19, Cys25, Gly66, Asp158, and Trp177 by hydrogen bonding them (Cathepsin L is lime green in the model, and the papain residues active in hydrogen bonding with Cathepsin L are highlighted with yellow halos). In addition to hydrogen bonding, hydrophobic interactions exist to exclude water, allowing the papain enzyme and Cathepsin L to associate even closer. Finally, between Trp177 and the Capthespin L molecule hold them tightly together.^[19] Cathepsin L plays a roll in many different diseases including malaria, leishmaniasis, Chagas' disease, African trypanosomiasis, toxoplasmosis, and amoebiasis. Some studies show that there is a relation between cathepsins and certain cancers, alzheimer's, and arthritis. ^[20]

Stefin B

acts as a competitive inhibitor to cysteine proteases-- it binds tightly but reversibly to the papain active site. Stefin inhibitors are characterized by M_r of about 11,000, no disulfide bonds and no associated carbohydrates.

In Stefin B, the Gly9 residue along with form a "wedge" complementary to the active site groove of papain. This wedge makes extensive and tight interactions with papain and a total of 128 intermolecular atom-atom interactions occur. Met6-Pro11, Gln53-Asn59, Gln101-His104 and Tyr124-Phe125 on the wedge all have some interaction to the enzyme though not always direct. All residues from the base and both sides of the are involved in the complex with the inhibtor (Trp177, Ser21, Cys63, Cys25, Asp158 and His159).

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. Fourteen of these bridges form a papain contact. The rest of the interactions are largely hydrophobic-- involving apolar . ^[21]