Biochemistry I Fall Term, 2000

November 6 & 8, 2000

Lectures 26 & 27: Enzyme Mechanism: Serine Proteases

Assigned reading in Campbell: Chapter 5.11-5.12

Key Terms:
Acid-base catalysis
Covalent catalysis
Acyl-enzyme intermediate
Nucleophilic agent
Electrophilic agent
Proximity and orientation effects
Preferential binding of transition state complex
Specificity constant (kcat/KM)
Catalytic triad
Rate limiting step (RLS)

Take the Review Quiz on Lecture 26 concepts.
The Review Quiz on Lecture 27 continues the topic.

Structure of a Trypsin-Inhibitor Complex: "Must-viewing" of an enzyme active site on a Chime page.
Serine Protease Mechanism: a JavaScript animation of the separate steps.
Serine Protease Mechanism: a (smoother) Shockwave animation of the separate steps.
Effects of pH on Enzyme Mechanism: a derivation and application to serine proteases.
A Catalytic Triad at High Resolution: JPEG of the Asp-His H-bond in a serine protease.
(11.8.00) The Peptidyl Transferase Reaction has similarities to the serine protease mechanism.


  • Serine proteases can hydrolyze either esters or peptide bonds utilizing mechanisms of covalent catalysis and preferential binding of the transition state.
  • Serine proteases play an important role in many processes, e.g. digestion of dietary protein, blood clotting cascade, and in several pathways of differentiation and development.
    Proteases active in digestion include:
    1. Trypsin
    2. Chymotrypsin
    3. Elastase
  • These enzymes are produced as zymogens. Proteolytic cleavage occurs in several locations resulting in the formation of intact enzymes. The active site in the zymogen is distorted and does not have high catalytic efficiency.
  • Each of the above enzymes has a different substrate specificity, to be discussed in more detail below.
  • The reaction involves the formation of a stable acyl-enzyme intermediate as shown by "burst kinetics" (i.e. the stoichiometric release of one of the products that is much faster than the steady state Vmax). For example, p-nitrophenol is rapidly released during the burst phase of p-nitrophenyl acetate hydrolysis.

Structural Features

Functional (active site) residues were identified in the following way:

Ser 195: reacts with diisopropylphosphofluoridate (DIPF can also be used as a nerve gas by inhibiting acetylcholinesterase)

His 57: modified by tosyl-L-phenylalanine chloromethyl ketone. (TPCK is specific for chymotrypsin.)

Asp 102: was identified only after a crystal structure had been determined.
Examination of homologous serine proteases show the following:

Crystal structures show that Ser 195, His 57, Asp 102 are close in space.

Ser 195, His 57, and Asp 102 are conserved (found in all serine proteases) and are called the catalytic triad.

The structure of the enzyme-inhibitor complex [trypsin-bovine trypsin inhibitor (BPTI)] shows the formation of a covalent complex between Ser 195 of trypsin and the scissile bond. In this complex the normal planar geometry of the peptide bond has been altered to a tetrahedral geometry. This allows the oxygen on the carboxyl group to occupy the oxyanion hole in the binding pocket. This in turn permits a favorable interaction of the oxygen with the amide groups of Ser 195 and Gly 193.

Proposed mechanism of trypsin cleavage:
(View the chemical structures in the two animations linked above.
Note: Campbell, Fig. 5.19 omits Asp 102 in this mechanism; the hydrogen on unprotonated His 57 should be on the other nitrogen.)

  1. Substrate binds.
  2. Nucleophilic attack of the side chain oxygen of Ser 195 on the carbonyl carbon of the scissile bond forming a tetrahedral intermediate. Assist from His 57 (proton transfer from Ser 195).
  3. Breakage of the peptide bond with assistance from His 57 (proton transfer to the new amino terminus).
  4. Release of the first product.
  5. Nucleophilic attack of water on the acyl-enzyme intermediate with assistance of His 57 and formation of the tetrahedral intermediate.
  6. Decomposition of acyl intermediate and release of the second product.

Nucleophile: a group that is electron rich and can form bonds with electron deficient groups. In this case the electronegativity of the oxygen makes the carbonyl carbon electron deficient.

Electrophile: a group that is electron poor and can form bonds with electron rich groups.

Role of Asp 102

  1. NMR studies show pKa of Asp 102 is very close to that of His 57.
  2. Formation of a very stable hydrogen bond that assists in delocalization of charge on His 57.

Chemical modification & mutagenesis studies

  1. Methylation of His 57 reduces activity by 104.
  2. Mutation or covalent modification of Ser 195, His 57, and Asp 102 reduce activity by 104.

Enhancement of Catalysis by Selective Binding to Transition State

According to transition state theory the rate contants of the uncatalyzed (kN) and catalyzed (kE) reactions are given by:

A 106 enhancement in rate implies about a 21 kJ/mol (5 kcal/mol) difference in free energy. This difference in free energy can be related to a difference in binding of the substrate versus the transition state.

Specificity of Three Proteases

Substrate specificity of:
  1. Elastase cleaves after small residues: No real binding pocket.
  2. Trypsin cleaves after Lys and Arg residues: Asp 189 interacts with the positive charge on Arg and Lys.
  3. Chymotrypsin cleaves after aromatic (and large hydrophobic) residues: Hydrophobic pocket shielded by Met 192.

kcat/KM as specificity constant: Review a portion of the Lecture 14 notes.

Steady-State Kinetics of Serine Proteases

Using the above scheme:
  1. If S is p-nitrophenyl acetate (Campbell, p. 181),
  2. P1 is p-nitrophenol;
  3. P2 is acetic acid.

Steps in the solution of the steady-state kinetics:
  1. Steady state assumption: d[EA]/dt = 0
  2. -k3[EA] + k2[ES] = 0
  3. KS = [E][S]/[ES]
  4. ET = [E] + [ES] + [EA]
  5. v = k3[EA]

The end result is:

Several Points:

  1. Although the velocity vs. [S] curves are hyperbolic, the Vmax and KM are complex functions of the various rate constants.
  2. The maximum velocity cannot be faster than the slowest, rate limiting step (RLS):
    • If k2 is fast then Vmax = k3.
    • If k3 is fast then Vmax = k2.

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