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1) triose phosphate isomerase
Catalyzes the interconversion of dihydroxyacetone phosphate (DHAP) and
glyceraldehyde 3-phosphate.
Enzyme has two ionizable active site residues Glu (glutamate)-165 and His-95--> act
as acid-base catalysts.
Mechanism:
1) Hydrogen bonds form between imidazole group of His-95 and carbonyl oxygen of
DHAP (COO- group is ionized; histidine active a.a.)
Carboxylate group of Glu-195 attacks proton of C-1 from substrate (DHAP) to
form enediolate intermediate.
2) C-2 oxygen (electron-rich) attacks proton of His-95 --> converts -O- to -OH to
form an enediol.
3) imidazole from of His-95 attacks -OH of C-1 to form another enediolate
intermediate
4) Glu-195 donates proton to C-2 --> glyceraldehyde 3-phosphate formed
2) lysozyme
Good example of transition state stabilization - enzyme binds to and stabilizes
transition state.
Action: hydrolyzes bacterial cell walls. Found in tears, saliva.
3
Substrate is alternating residues of N-acetylglucosamine (GlcNAc) and Nacetylmuramic
acid (MurNAc).
Enzyme hydrolyzes glycosidic bond.
Binding site can hold 6 sugars or residues labeled A, B, C, D, E, F - one of the
residues (D) must be bent into half-chair conformation.
Glu-35 and Asp-52 are the two a.m. residues most involved.
Mechansism:
1) MurNAc residues bind at B, D, F --> residue D turned into half-chair
conformation.
2) Glu-35 acts as acid catalyst --> donates H+ to O of glycosidic bond between D and
E --> glycosidic bond broken
3) Residues bound in E and F diffuse out of binding site.
4) Replaced by a molecule of water.
5) C-1 now is a carbonium ion intermediate C+.
6) Asp-52 stabilizes carbonium intermediate via charge-charge interactions.
7) Glu-35 O- attacks H+ from water molecule --> OH- is added to C+ ion
8) R-MurNAc diffuses away (enzyme has been reprotonated; no charges to hold it in
place).
3- chymotrypsin, a member of the serine proteases
Examples: trypsin, chymotrypsin, and elastase that catalyze much of the digestion in the
small intestine.
Synthesized in pancreas; stored as inactive precursors called zymogens - prevents damage
to cell.
What is actually released from the cell is trypsinogen, chymotrypsinogen, proelastase, which
are then activated by selective proteolysis.
Enzyme called enteropeptidase activates trypsinogen --> trypsin by cleaving of N-terminal
hexapeptide.
Trypsin then activates the other two.
4
All three enzymes have similar primary, second, tertiary structure.
All cleave peptide bonds on COOH side of hydrophobic or aromatic side chains.
Substrate specificity is due to amino acid residues in the hydrophobic binding pocket.
chymotrypsin - serine (uncharged) --> accepts large, bulky, hydrophobic
side groups.
trypsin - aspartate ( negatively charged) accepts Lys, Arg, Gly, Ala.
elastase - shallow - binds a.a. with small side chains ( Gly, Ala).
Catalysis involves use of catalytic triad:
Ser-195 His-57 Asp-102
All three amino acids are hydrogen bonded.
Ser-195 residue is highly reactive; very unusual.
Mechansim of chymotrypsin:
1) Substrate enters enzyme and is aligned with R1 group in binding pocket --> places
carbonyl carbon of peptide bond next to oxygen of Ser- 195.
2) His-57 attacks H of Ser-195.
3) Now, the nucleophilic oxygen of Ser-195 attacks carbonyl carbon of peptide bond to
form tetrahedral intermediate (transition state?).
4) C=O bonds changes to a single bond (oxygen is negatively charged = oxyanion) and
forms H-bond with -NH groups of Gly-193 and Ser-195.
5) His-57 and Asp-102 share H+ in low-barrier hydrogen bond (increases rate of
catalysis by decreasing activation energy; very strong hydrogen bond).
6) His-57 imidazolium ring acts as an acid catalyst by donating H to peptide bond -->
molecule cleaved --> amine product released.
7) Carbonyl group of peptide forms covalent bond with enzyme --> acyl-enzyme
intermediate formed.
8) After first product leaves, a molecule of water enters --> donates H+ to His-57 --> -
OH group left attacks carbonyl group --> formation of second tetrahedral
intermediate and stabilized by oxyanion hole plus a low barrier H-bond.
9) His-57 donates a proton --> second intermediate collapses
10) Second product is formed, released from active site --> chymotrypsin regenerated.
Shows: covalent catalysis (Ser), acid-base catalysis (His)
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