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
Human Heredity: Principles and Issues (MindTap Course List)
11th Edition
ISBN:9781305251052
Author:Michael Cummings
Publisher:Michael Cummings
Chapter10: From Proteins To Phenotypes
Section: Chapter Questions
Problem 9QP: a. Compounds A, B, C, and D are known to be intermediates in the pathway for production of protein...
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