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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
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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 nutant enzymes are shown in the table. NH—CH-COO T CH₂ COO™ Aspartate α-Amino 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) Asp-AT a-Keto acid 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.
Transcribed Image Text: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 nutant enzymes are shown in the table. NH—CH-COO T CH₂ COO™ Aspartate α-Amino 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) Asp-AT a-Keto acid 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.
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