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(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 (EGFR988-998). 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? Substrate DADEPYLIPQQG DADAPYLIPQQG DAAEPYLIPQQG AAAAPYLIPQQ G kcat 44.6 1.8 39.8 ± 0.32 35.3 ± 0.22 34.7 ± 0.25 A(-3) D(-2) Tyr46 Ser216 Arg47 E(-1) Asp181 PTP1B Km μM 3.9 ± 0.9 13.7 ± 0.46 52.7 ± 0.7 6.6 ± 0.22 Y(0) Asp48 L(+1) Phe 182 Gln262 kcat/Km 10-7 X (S¹M¹) 1.1 ± 0.25 0.29 ± 0.01 0.53 ± 0.02 0.066 0.001

(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 (EGFR988-998). 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? Substrate DADEPYLIPQQG DADAPYLIPQQG DAAEPYLIPQQG AAAAPYLIPQQ G kcat 44.6 1.8 39.8 ± 0.32 35.3 ± 0.22 34.7 ± 0.25 A(-3) D(-2) Tyr46 Ser216 Arg47 E(-1) Asp181 PTP1B Km μM 3.9 ± 0.9 13.7 ± 0.46 52.7 ± 0.7 6.6 ± 0.22 Y(0) Asp48 L(+1) Phe 182 Gln262 kcat/Km 10-7 X (S¹M¹) 1.1 ± 0.25 0.29 ± 0.01 0.53 ± 0.02 0.066 0.001

Biochemistry
Biochemistry
6th Edition
ISBN:9781305577206
Author:Reginald H. Garrett, Charles M. Grisham
Publisher:Reginald H. Garrett, Charles M. Grisham
Chapter31: Completing The Protein Life Cycle: Folding, Processing, And Degradation
Section: Chapter Questions
Problem 16P
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To identify the critical enzyme resi- (c) ( due:substrate interaction and determinant of sub- 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. L(+1) Тyr46 Gln262 In the adjacent diagram the structural interactions between active site residues are illustrated for the Y(0) 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? Phe 182 Asp181 PTP1Β Substrate kcat kcat/Km UM 10-7 x (s-1 M1) DADEPYLIPQQG DADAPYLIPQG 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 0.53 + 0.02 35.3 + 0.22 6.6 + 0.22 34.7 + 0.25 52.7 + 0.7 0.066 + 0.001
Transcribed Image Text:To identify the critical enzyme resi- (c) ( due:substrate interaction and determinant of sub- 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. L(+1) Тyr46 Gln262 In the adjacent diagram the structural interactions between active site residues are illustrated for the Y(0) 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? Phe 182 Asp181 PTP1Β Substrate kcat kcat/Km UM 10-7 x (s-1 M1) DADEPYLIPQQG DADAPYLIPQG 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 0.53 + 0.02 35.3 + 0.22 6.6 + 0.22 34.7 + 0.25 52.7 + 0.7 0.066 + 0.001
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