Bio Chem 131

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Proteinases (Proteases)

Cleave proteins Four Families-based on functional group in the active site Serine Cysteine • Aspartic • Metallo

Serine Proteinases • Chymotrypsin--mammalian digestive enzyme which cleaves tryptophan, tyrosine, phenylalanine, and methionine • How do proteinases work?

How do serine proteinases cleave peptide bonds

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proteinases allow this reaction to occur? Four important structural features of serine proteinase – Catalytic triad – Oxyanion hole – Nonspecific bonding – Specificity pocket

CHYMOTRYPSIN • Two domains • – ~120 a. a. each • – Antiparallel betabarrel

Carboxypeptidase •

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A peptide substrate binds at the active site of the enzyme. X-ray structures of the enzyme with and without a competitive inhibitor show a large conformational change at the active site when inhibitor or substrate is bound. Without inhibitor, several waters occupy the active site. When inhibitor and presumably substrate are bound, the water leaves (which is entropically favored), and Tyr 248 swings around from near the surface of the protein in the absence of a molecule in the active site to interact with the carboxyl group of the bound molecule, a distance of motion equal to about 1/4 the diameter of the protein. This effectively closes off the active site and expels the water. A Zn2+ ion is present at the active site. It is bound by His 69, His 196, Glu 72, and finally a water molecule as the fourth ligand. A hydrophobic pocket which interacts with the phenolic group of the substrate accounts for the specificity of the protein. In the catalytic mechanism, Zn2+ helps polarize the labile amide bond, while Glu 270, acting as a general base, which along with Zn2+ helps promote dissociation of a proton from the bound water, making it a better nucleophile. Water attacks the elctrophilic carbon of the sessile bond, with Glu 270 acting as a general base catalyst. The tetrahedral intermediate then collapses, expelling the leaving amine group, which picks up a proton from Glu 270, which now acts as a general acid catalyst. People used to believe that Tyr 248 acted as a general acid, but mutagenesis showed that Tyr 248 can be replaced with Phe 248 without significant effect on the rate of the reaction.

CarboxypeptidaseA

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• Carboxypeptidase A (CPA) hydrolyzes C-terminal amino acids of polypeptide chains, exhibiting a preference towards substrates with large hydrophobic side chains (such as Phe) – metalloexopeptidase with 1 mole Zn2+ per mole enzyme – 307 amino acids, 34.5 kDa – excellent crystal structure of free form (1.54 Å resolution) • also structures of substrate analogs, TS analogs, slowly hydrolyzed • substrates, and product analogs complexed with CPA • – Zn2+ coordinated by Glu-72, His-69, His-196, and H2O

Mechanism for CPA • • • • • • • • • • • • • •

Possible mechanisms for CPA • 2 possible mechanisms were proposed for the role of Glu270 – Activated water mechanism: (Glu-270 as a general base) Glu270, perhaps in conjunction with Zn2+, activates water as a nucleophile to hydrolyze the peptide bond – Nucleophilic mechanism: (Glu-270 as a nucleophile) Glu-270 acts as a nucleophile directly to hydrolyze peptide bonds, forming an anhydride intermediate • The crystal structure of CPA does not distinguish between these two mechanisms (controversial whether Glu-270 is close enough for nucleophilic attack) – 18O isotope labeling experiments helped to determine peptide hydrolysis mechanism (reverse reactions with 18O labeled acid)

CPA Mechanism

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The carbonyl that is H-bonded to Arg127 is polarized Zn2+-bound H2O can act as a nucleophile, Glu-270 acts as general base tetrahedral intermediate is stabilized by Zn2+– aldehyde and ketone analogs are found in their hydrated forms in CPA crystal structures • Tyr-248 or Glu-270 act as a general acid to protonate leaving group amine

Lysozyme •

This enzyme, found in cells and secretions of vertebrates but also in viruses which infect bacteria, cleaves peptidoglycan GlcNAc (b 1->4) MurNAc repeat linkages (NAG-NAM) in the cell walls of bacteria and the GlcNAc (b 1->4) GlcNAc (poly-NAG) in chitin, found in the cells walls of certain fungi. Since these polymers are hydrophilic, the active site of the enzyme would be expected to contain a solvent-accessible channel into which the polymer could bind. The crystal structures of lysozyme and complexes of lysozyme and NAG have been solved to high resolution. The inhibitors and substrates form strong H bonds and some hydrophobic interactions with the enzyme cleft. Kinetic studies using (NAG)n polymers show a sharp increase in kcat as n increases from 4 to 5. The kcat for NAG6 and (NAGNAM)3 are similar. Models studies have shown that for catalysis to occur, (NAG-NAM)3 binds to the active site with each sugar in the chair conformation except the fourth which is distorted to a half chair form, which labilizes the glycosidic link between the 4th and 5th sugars. Additional studies show that if the sugars that fit into the binding site are labeled A-F, then because of the bulky lactyl substituent on the NAM, residues C and E can not be NAM, which suggests that B, D and F must be NAM residues. Cleavage occurs between residues D and E.

Lysozyme •

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Catalysis by the enzyme involves Glu 35 and Asp 52 which are in the active site. Asp 52 is surrounded by polar groups but Glu 35 is in a hydrophobic environment. This should increase the apparent pKa of Glu 35, making it less likely to donate a proton and acquire a negative charge at low pH values, making it a better general acid at higher pH values. The general mechanism appears to involve: binding of a hexasaccharide unit of the peptidoglycan with concomitant distortion of the D NAM. protonation of the sessile acetal O by the general acid Glu 35 (with the elevated pKa), which facilitates cleavage of the glycosidic link and formation of the resonant stabilized oxonium ion. Asp 52 stabilizes the positive oxonium through electrostatic catalysis. The distorted half-chair form of the D NAM stabilizes the oxonium which requires co-planarity of the substituents attached to the sp2 hybridized carbon of the carbocation resonant form (much like we saw with the planar peptide bond). water attacks the stablized carbocation, forming the hemiacetal with release of the extra proton from water to the deprotonated Glu 35 reforming the general acid catalysis.

Lysozyme • Binding and distortion of the D substituent of the substrate (to the half chair form as shown above) occurs before catalysis. Since this distortion helps stabilize the oxonium ion intermediate, it presumably stabilizes the transition state as well. Hence this enzyme appears to bind the transition state more tightly than the free, undistorted substrate, which is yet another method of catalysis. • pH studies show that side chains with pKa's of 3.5 and 6.3 are required for activity. These presumably correspond to Asp 52 and Glu 35, respectively. If the carboxy groups of lysozyme are chemically modified in the presence of a competitive inhibitor of the enzyme, the only protected carboxy groups are Asp 52 and Glu 35.