Question

1. The HIV protease structure was solved very early in the process of developing anti-HIV drugs. Importantly, the protease was found to be quite similar to proteases like chymotrypsin and trypsin. Compare and contrast the catalytic mechanism and binding properties of chymotrypsin, trypsin, and the HIV protease.

Answer 1

chymotrypsin

Chymotrypsin, a protease, is an enzyme that cleaves the carbonyl side of certain peptide bonds by both general acid-base catalysis, but primarily covalent catalysis. In this mechanism, a nucleophile becomes covalently attached to a substrate in a transition state with an acyl-enzyme. The protease cleaves proteins by a hydrolysis reaction, an addition of a water molecule. The double bond between the carbon and nitrogen strengthens its bond. Chymotrypsin is site specific and will only cleave the carboxyl side of large hydrophobic or aromatic amino acids such as phenylalanine (Phe), methionine (Met), tyrosine (Tyr), and tryptophan (Trp), unless the next amino acid is proline (Pro). The reason why chymotrypsin prefers to cleave specifically to bulky hydrophobic amino acids is due to the formation of S1 pockets,which, in the case of chymotrypsin, is lined with relatively hydrophobic residues such as Ser-189, Ser-214, Trp-215, Gly-216, and Gly-226. Chymotrypsin catalyzes the reaction rate by a factor of 109. The reaction has two steps, an acylation phase and a deacylation phase. In the former phase, the peptide bond is cleaved and an ester is formed between substrate and enzyme. In the latter phase, this ester is hydrolyzed and the enzyme is regenerated.

Mechanism of Chymotrypsin

V - HN Car-NH Asp 102 His 57 Ser 105

The triad is H-bonded: Asp is H-bonded to His to keep it oriented. His is H-bonded to Ser. This increases the nucleophilicity of the Ser oxygen. His acts as a general base, abstracting a proton from Ser Ser attacks the carbonyl carbon of the peptide bond of the substrate. Forms first tetrahedral intermediate His now acts as a general acid by transferring a proton to nitrogen of first tetrahedral intermediate This causes the peptide bond to break, and the first product leaves.The acyl enzyme remains. This is a covalent intermediate in which the Ser is esterified His now acts as a general base- activates attack of the Ser ester by water OH. Second tetrahedral intermediate forms His transfers its proton back to Ser to break down intermediate The second product is released- The original enzyme is regenerated

trypsin

trypsinis a serine protease from the PA clan superfamily, found in the digestive system of many vertebrates, where it hydrolyzes proteins. Trypsin is formed in the small intestine when its proenzyme form, the trypsinogen produced by the pancreas, is activated. Trypsin cleaves peptide chains mainly at the carboxyl side of the amino acids lysine or arginine. It is used for numerous biotechnological processes. The process is commonly referred to as trypsin proteolysis or trypsinisation, and proteins that have been digested/treated with trypsin are said to have been trypsinized. Trypsin was discovered in 1876 by Wilhelm Kühne and was named from the Ancient Greek word for rubbing since it was first isolated by rubbing the pancreas with glycerin.

Mechanism

The enzymatic mechanism is similar to that of other serine proteases. These enzymes contain a catalytic triad consisting of histidine-57, aspartate-102, and serine-195. This catalytic triad was formerly called a charge relay system, implying the abstraction of protons from serine to histidine and from histidine to aspartate, but owing to evidence provided by NMR that the resultant alkoxide form of serine would have a much stronger pull on the proton than does the imidazole ring of histidine, current thinking holds instead that serine and histidine each have effectively equal share of the proton, forming short low-barrier hydrogen bonds therewith. By these means, the nucleophilicity of the active site serine is increased, facilitating its attack on the amide carbon during proteolysis. The enzymatic reaction that trypsin catalyzes is thermodynamically favorable, but requires significant activation energy (it is "kinetically unfavorable"). In addition, trypsin contains an "oxyanion hole" formed by the backbone amide hydrogen atoms of Gly-193 and Ser-195, which through hydrogen bonding stabilize the negative charge which accumulates on the amide oxygen after nucleophilic attack on the planar amide carbon by the serine oxygen causes that carbon to assume a tetrahedral geometry. Such stabilisation of this tetrahedral intermediate helps to reduce the energy barrier of its formation and is concomitant with a lowering of the free energy of the transition state. Preferential binding of the transition state is a key feature of enzyme chemistry.

The negative aspartate residue (Asp 189) located in the catalytic pocket (S1) of trypsin is responsible for attracting and stabilizing positively charged lysine and/or arginine, and is, thus, responsible for the specificity of the enzyme. This means that trypsin predominantly cleaves proteins at the carboxyl side (or "C-terminal side") of the amino acids lysine and arginine except when either is bound to a C-terminal proline, although large-scale mass spectrometry data suggest cleavage occurs even with proline. Trypsin is considered an endopeptidase, i.e., the cleavage occurs within the polypeptide chain rather than at the terminal amino acids located at the ends of polypeptides.

HIV protease

HIV-1 protease (PR) is a retroviral aspartyl protease (retropepsin), an enzyme involved with peptide bond hydrolysis in retroviruses, that is essential for the life-cycle of HIV, the retrovirus that causes AIDS.HIV protease cleaves newly synthesized polyproteins (namely, Gag and Gag-Pol) at nine cleavage sites to create the mature protein components of an HIV virion, the infectious form of a virus outside of the host cell.[4] Without effective HIV protease, HIV virions remain uninfectious.

mechanism

As an aspartic protease, the dimerized HIV-1 PR functions through the aspartyl group complex, in order to perform hydrolysis. Of the two Asp25 residues on the combined catalytic active site of HIV-1 PR, one is deprotonated while the other is protonated, due to pKa differences from the micro-environment.

In a general aspartic protease mechanism, once the substrate is properly bound to the active site of the enzyme, the deprotonated Asp25 catalytic amino acid undergoes base catalysis, rendering the incoming water molecule a better nucleophile by deprotonating it. The resulting hydroxyl ion attacks the carbonyl carbon of the peptide bond, forming an intermediate with a transient oxyanion, which is stabilized by the initially protonated Asp25. The oxyanion re-forms a double bond, leading to the cleavage of the peptide bond between the two amino acids, while the initially deprotonated Asp25 undergoes acid catalysis to donate its proton to the amino group, making the amino group a better leaving group for complete peptide bond cleavage and returning to its original deprotonated state.

While HIV-1 PR shares many of the same characteristics as a non-viral aspartic protease, some evidence has shown that HIV-1 PR catalyzes hydrolysis in a concerted manner; in other words, the nucleophilic water molecule and the protonated Asp25 simultaneously attack the scissile peptide bond during catalysis