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Asp48 E(-1) D(-2) H (c) ( To identify the critical enzyme resi- due:substrate interaction and determinant of sub- strate specificity, steady-state kinetic studies have D(-4) been carried out by site-specific mutagenesis of the enzyme and by systematically changing amino acid residues in the 11-amino acid phosphotyrosine con- taining substrate that simulate residues 988 - 998 of the epidermal growth factor receptor (EGFR 988-998). The table below compares the kinetic parameters of the wild type enzyme with phosphotyrosine pep- tides in which amino acid residues are substituted. In the adjacent diagram the structural interactions between active site residues are illustrated for the first 6 residues of the substrate. Identify which sub- strate residue is most sensitive to substitution and justify your conclusion on the basis of the kinetic parameters. Is the decrease in Kcat/ KM due to loss of catalytic reactivity or due to suboptimal position- ing of the phospho-Tyr residue in the active site? A(-3) Tyr46 Ser216 Arg47 Asp181 Substrate DADEpYLIPQQG DADAP YLIPQQG DAAEpYLIPQQG AAAAP YLIPQQG kcat 5-1 44.6 ± 1.8 39.8 ± 0.32 35.3 ± 0.22 34.7 ± 0.25 PTP1B Km Y(0) L(+1) Gln262 Phe 182 kcat/Km им 3.9 ± 0.9 13.7 ± 0.46 6.6 ± 0.22 52.7 ± 0.7 10-7 × (s¯¹ M¯¹) 1.1 ± 0.25 0.29 ± 0.01 0.53 ± 0.02 0.066 ± 0.001

Asp48 E(-1) D(-2) H (c) ( To identify the critical enzyme resi- due:substrate interaction and determinant of sub- strate specificity, steady-state kinetic studies have D(-4) been carried out by site-specific mutagenesis of the enzyme and by systematically changing amino acid residues in the 11-amino acid phosphotyrosine con- taining substrate that simulate residues 988 - 998 of the epidermal growth factor receptor (EGFR 988-998). The table below compares the kinetic parameters of the wild type enzyme with phosphotyrosine pep- tides in which amino acid residues are substituted. In the adjacent diagram the structural interactions between active site residues are illustrated for the first 6 residues of the substrate. Identify which sub- strate residue is most sensitive to substitution and justify your conclusion on the basis of the kinetic parameters. Is the decrease in Kcat/ KM due to loss of catalytic reactivity or due to suboptimal position- ing of the phospho-Tyr residue in the active site? A(-3) Tyr46 Ser216 Arg47 Asp181 Substrate DADEpYLIPQQG DADAP YLIPQQG DAAEpYLIPQQG AAAAP YLIPQQG kcat 5-1 44.6 ± 1.8 39.8 ± 0.32 35.3 ± 0.22 34.7 ± 0.25 PTP1B Km Y(0) L(+1) Gln262 Phe 182 kcat/Km им 3.9 ± 0.9 13.7 ± 0.46 6.6 ± 0.22 52.7 ± 0.7 10-7 × (s¯¹ M¯¹) 1.1 ± 0.25 0.29 ± 0.01 0.53 ± 0.02 0.066 ± 0.001

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
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(c) ( due:substrate interaction and determinant of sub- To identify the critical enzyme resi- D(-2) Arg47 strate specificity, steady-state kinetic studies have D(-4). been carried out by site-specific mutagenesis of the enzyme and by systematically changing amino acid residues in the 11-amino acid phosphotyrosine con- taining substrate that simulate residues 988 – 998 of the epidermal growth factor receptor (EGFR988–998). Asp48 E(-1) A(-3) The table below compares the kinetic parameters of the wild type enzyme with phosphotyrosine pep- tides in which amino acid residues are substituted. In the adjacent diagram the structural interactions L(+1) Tyr46 Gln262 Y(0) between active site residues are illustrated for the first 6 residues of the substrate. Identify which sub- strate residue is most sensitive to substitution and Ser216 justify your conclusion on the basis of the kinetic parameters. Is the decrease in kcat/ KM due to loss of catalytic reactivity or due to suboptimal position- ing of the phospho-Tyr residue in the active site? Phe182 Asp181 PTP1B Substrate kcat K. kcat/Km JUM 10-7 x (s-1 M1) DADEPYLIPQQG DADAPYLIPQQG DAAEPYLIPQQG AAAAPYLIPQQG 44.6 ± 1.8 39.8 + 0.32 3.9 + 0.9 13.7 + 0.46 1.1 + 0.25 0.29 + 0.01 35.3 + 0.22 6.6 ± 0.22 0.53 + 0.02 34.7 + 0.25 52.7 + 0.7 0.066 0.001
Transcribed Image Text:(c) ( due:substrate interaction and determinant of sub- To identify the critical enzyme resi- D(-2) Arg47 strate specificity, steady-state kinetic studies have D(-4). been carried out by site-specific mutagenesis of the enzyme and by systematically changing amino acid residues in the 11-amino acid phosphotyrosine con- taining substrate that simulate residues 988 – 998 of the epidermal growth factor receptor (EGFR988–998). Asp48 E(-1) A(-3) The table below compares the kinetic parameters of the wild type enzyme with phosphotyrosine pep- tides in which amino acid residues are substituted. In the adjacent diagram the structural interactions L(+1) Tyr46 Gln262 Y(0) between active site residues are illustrated for the first 6 residues of the substrate. Identify which sub- strate residue is most sensitive to substitution and Ser216 justify your conclusion on the basis of the kinetic parameters. Is the decrease in kcat/ KM due to loss of catalytic reactivity or due to suboptimal position- ing of the phospho-Tyr residue in the active site? Phe182 Asp181 PTP1B Substrate kcat K. kcat/Km JUM 10-7 x (s-1 M1) DADEPYLIPQQG DADAPYLIPQQG DAAEPYLIPQQG AAAAPYLIPQQG 44.6 ± 1.8 39.8 + 0.32 3.9 + 0.9 13.7 + 0.46 1.1 + 0.25 0.29 + 0.01 35.3 + 0.22 6.6 ± 0.22 0.53 + 0.02 34.7 + 0.25 52.7 + 0.7 0.066 0.001
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