BCH 261 Lecture Notes - Lecture 11: Chief Operating Officer, Hexokinase, Enzyme Kinetics
7 Jun 2018
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Page 1 of 8
Ryerson University – Winter 2018
BCH 261-011&071 Biochemistry -April 4th, 2018
BCH 261-011&071 Biochemistry -April 4th, 2018
Lecture
Structure and Catalytic mechanism of chymotrypsin
Chymotrypsin is an enzyme active in the intestinal tract – produced and released by the
pancreas. It catalyzes the hydrolysis of proteins, degrading them into smaller molecules called
peptides. Peptides are further split into free amino acids.
Asp102
● Positions the histidine
● Tight hydrogen bond between oxygen and proton
His57
● Catalytic base
● Nitrogen wants to be protons red and act like a base
● Nitrogen takes portion from OH of serine
Chymotrypsin mechanism
Formation of covalent intermediate
● Polypeptide substrate binds noncovalently in enzyme active site
● Catalytic triad include reactive serine nucleophile that attacks electrophilic amide C atom
● Resulting tetrahedral oxyanion is stabilized by hydrogen bond interaction with oxyanion hole
● Collapse of intermediate and hydrogen ion transfer from His57 leads to cleavage of C-N bond
● N-terminal peptide is bound through acyl linkage to serine
Page 2 of 8
Ryerson University – Winter 2018
BCH 261-011&071 Biochemistry -April 4th, 2018
Second covalent intermediate
● Water molecule binds to active site and attracts acts ester carbonyl
● Resulting tetrahedral oxyanion intermediate is stabilized via enthalpic interaction with
oxyanion hope
● Second peptide fragment is released and the enzyme return to initial state
Questions you should be able to answer in regards to chymotrypsin mechanism:
*Do not memorize steps, you need to know chemical logic
● What is the nucleophile?
● What other amino acids can fill that role?
● How is the nucleophile activated?
● Role of His57 and Asp102
● What is the acyl-enzyme intermediate?
● How is product released from the intermediate?
Enzyme Kinetics
Kinetics
● The rates of chemical or biochemical reaction
Rate of enzyme reaction is affected by:
● Enzyme concentration
● Substrate concentration
● Temperature
● Effectors
● pH
Why study kinetics?
Quantitative description of bio catalysis
● Determining order of binding substrates
● Elucidate acid base catalysis
● Catalytic mechanism
Page 3 of 8
Ryerson University – Winter 2018
BCH 261-011&071 Biochemistry -April 4th, 2018
Basic kinetic equation
E + S ⇌ ES (kcat)⇌ E + P
● E = enzyme
● S = substrate
● ES = enzyme substrate complex
● P = product
V = kcat [ES]
● V = reaction rate
ES is essentially constant so it is referred to as steady state
Steady state kinetics
● Studies initial rate V0
● At the beginning , substrate assumed to be constant so amount of product is small
● ES → E + P is the rate limiting step
● Overall rate is proportional to ES
Michaelis-Menten equation - model of enzyme kinetics
describes the rate of enzymatic reactions
by relating reaction rate V (rate of formation of product P) to [S] (the concentration of substrate S)
Equation:
V = Vmax[S]/Km + [S]
Km = [S][E]/[ES]
Vmax represents the maximum rate achieved by the system, at saturating substrate concentration.
The Michaelis constant Km is the substrate concentration
at which the reaction rate is half of Vmax
Document Summary
Chymotrypsin is an enzyme active in the intestinal tract produced and released by the pancreas. It catalyzes the hydrolysis of proteins, degrading them into smaller molecules called peptides. Peptides are further split into free amino acids. Tight hydrogen bond between oxygen and proton. Nitrogen wants to be protons red and act like a base. Nitrogen takes portion from oh of serine. Polypeptide substrate binds noncovalently in enzyme active site. Catalytic triad include reactive serine nucleophile that attacks electrophilic amide c atom. Resulting tetrahedral oxyanion is stabilized by hydrogen bond interaction with oxyanion hole. Collapse of intermediate and hydrogen ion transfer from his57 leads to cleavage of c-n bond. N-terminal peptide is bound through acyl linkage to serine. Water molecule binds to active site and attracts acts ester carbonyl. Resulting tetrahedral oxyanion intermediate is stabilized via enthalpic interaction with oxyanion hope. Second peptide fragment is released and the enzyme return to initial state.
