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3. Aspartate aminotransferase (AspAT) catalyzes the following reaction: COO™ C=O CH₂ COO Oxaloacetate The AspAT enzyme has two active-site arginines, Arg 386 and Arg 292, that interact with the a- carboxylate and ß-carboxylate groups on the aspartate substrate, respectively. Investigators stud- ied the mechanism of AspAT in more detail by constructing mutant AspAT enzymes in which either or both of the essential arginines were replaced with a lysine residue. The kinetic parame- ters for the wild-type enzyme and mutant enzymes are shown in the table. NH—CH-COO T CH₂ T COO™ Aspartate a-Amino acid Asp-AT a-Keto acid Enzyme 1. Wild-type Asp AT(Arg 292 Arg 386) 2. Mutant Asp AT(Lys 292 Arg 386) 3. Mutant Asp AT(Arg 292 Lys 386) 4. Mutant Asp AT(Lys 292 Lys 386) KM (mm) 4 326 72 300 Kcat (S-1) 530 4.5 9.6 0.055 (a) The Michaelis constant is strictly not equivalent to an equilibrium binding constant; however under certain conditions the Michaelis constant does approach the equilibrium (dissoci- ation) constant for substrate binding to the enzyme. State the condition and assume that it holds for the AspAT enzyme and mutants and compare the capacity of the aspartate substrate to bind to the wild-type and mutant enzymes. (b) What kinetic parameter is used to evaluate the catalytic efficiency of an enzyme and to what condition of the enzyme/substrate reaction does it apply? Evaluate the catalytic efficiency of the wild-type and mutant enzymes.

3. Aspartate aminotransferase (AspAT) catalyzes the following reaction: COO™ C=O CH₂ COO Oxaloacetate The AspAT enzyme has two active-site arginines, Arg 386 and Arg 292, that interact with the a- carboxylate and ß-carboxylate groups on the aspartate substrate, respectively. Investigators stud- ied the mechanism of AspAT in more detail by constructing mutant AspAT enzymes in which either or both of the essential arginines were replaced with a lysine residue. The kinetic parame- ters for the wild-type enzyme and mutant enzymes are shown in the table. NH—CH-COO T CH₂ T COO™ Aspartate a-Amino acid Asp-AT a-Keto acid Enzyme 1. Wild-type Asp AT(Arg 292 Arg 386) 2. Mutant Asp AT(Lys 292 Arg 386) 3. Mutant Asp AT(Arg 292 Lys 386) 4. Mutant Asp AT(Lys 292 Lys 386) KM (mm) 4 326 72 300 Kcat (S-1) 530 4.5 9.6 0.055 (a) The Michaelis constant is strictly not equivalent to an equilibrium binding constant; however under certain conditions the Michaelis constant does approach the equilibrium (dissoci- ation) constant for substrate binding to the enzyme. State the condition and assume that it holds for the AspAT enzyme and mutants and compare the capacity of the aspartate substrate to bind to the wild-type and mutant enzymes. (b) What kinetic parameter is used to evaluate the catalytic efficiency of an enzyme and to what condition of the enzyme/substrate reaction does it apply? Evaluate the catalytic efficiency of the wild-type and mutant enzymes.

Biochemistry
Biochemistry
9th Edition
ISBN:9781319114671
Author:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Publisher:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Chapter1: Biochemistry: An Evolving Science
Section: Chapter Questions
Problem 1P
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**Aspartate aminotransferase (AspAT) Reaction and Kinetics** **Reaction:** Aspartate aminotransferase (AspAT) catalyzes the transfer of an amino group from an α-amino acid to an α-keto acid, resulting in the formation of a new α-amino acid and α-keto acid. The reaction involves: - Aspartate as a substrate, which gets converted to oxaloacetate. - An α-amino acid that becomes an α-keto acid after the reaction. The schematic diagram illustrates this chemical reaction. **Enzyme Details:** The AspAT enzyme has two active-site arginines, Arg 386 and Arg 292, which interact with the α-carboxylate and β-carboxylate groups on the aspartate substrate, respectively. Researchers studied AspAT by creating mutants wherein these arginines were substituted with lysine residues. The kinetic parameters for both wild-type and mutant enzymes are listed in the table. **Kinetic Parameters:** | Enzyme | \( K_M \) (mM) | \( k_{\text{cat}} \) (s\(^{-1}\)) | |----------------------------------------|---------------|----------------------------------| | 1. Wild-type Asp AT(Arg 292 Arg 386) | 4 | 530 | | 2. Mutant Asp AT(Lys 292 Arg 386) | 326 | 4.5 | | 3. Mutant Asp AT(Arg 292 Lys 386) | 72 | 9.6 | | 4. Mutant Asp AT(Lys 292 Lys 386) | 300 | 0.055 | **Questions:** (a) The Michaelis constant (\( K_M \)) is not strictly an equilibrium binding constant but can approximate the equilibrium constant under certain conditions. Identify these conditions and assume they are applicable to AspAT and its mutants. Compare the substrate binding capacity of the wild-type and mutant enzymes using \( K_M \) values. (b) Identify the kinetic parameter used to evaluate the catalytic efficiency of an enzyme. Discuss the enzyme/substrate conditions for which it applies. Evaluate the catalytic efficiency of the wild-type and mutant enzymes using \( k_{\text{cat}}/K_M \). **Graph/Diagram Explanation:** The diagram shows the conversion of an α-amino acid (as
Transcribed Image Text:**Aspartate aminotransferase (AspAT) Reaction and Kinetics** **Reaction:** Aspartate aminotransferase (AspAT) catalyzes the transfer of an amino group from an α-amino acid to an α-keto acid, resulting in the formation of a new α-amino acid and α-keto acid. The reaction involves: - Aspartate as a substrate, which gets converted to oxaloacetate. - An α-amino acid that becomes an α-keto acid after the reaction. The schematic diagram illustrates this chemical reaction. **Enzyme Details:** The AspAT enzyme has two active-site arginines, Arg 386 and Arg 292, which interact with the α-carboxylate and β-carboxylate groups on the aspartate substrate, respectively. Researchers studied AspAT by creating mutants wherein these arginines were substituted with lysine residues. The kinetic parameters for both wild-type and mutant enzymes are listed in the table. **Kinetic Parameters:** | Enzyme | \( K_M \) (mM) | \( k_{\text{cat}} \) (s\(^{-1}\)) | |----------------------------------------|---------------|----------------------------------| | 1. Wild-type Asp AT(Arg 292 Arg 386) | 4 | 530 | | 2. Mutant Asp AT(Lys 292 Arg 386) | 326 | 4.5 | | 3. Mutant Asp AT(Arg 292 Lys 386) | 72 | 9.6 | | 4. Mutant Asp AT(Lys 292 Lys 386) | 300 | 0.055 | **Questions:** (a) The Michaelis constant (\( K_M \)) is not strictly an equilibrium binding constant but can approximate the equilibrium constant under certain conditions. Identify these conditions and assume they are applicable to AspAT and its mutants. Compare the substrate binding capacity of the wild-type and mutant enzymes using \( K_M \) values. (b) Identify the kinetic parameter used to evaluate the catalytic efficiency of an enzyme. Discuss the enzyme/substrate conditions for which it applies. Evaluate the catalytic efficiency of the wild-type and mutant enzymes using \( k_{\text{cat}}/K_M \). **Graph/Diagram Explanation:** The diagram shows the conversion of an α-amino acid (as
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