To identify the critical enzyme resi-(c) (due:substrate interaction and determinant of sub-D(-2)Arg47strate specificity, steady-state kinetic studies have D(-4)been carried out by site-specific mutagenesis of theenzyme and by systematically changing amino acidresidues in the 11-amino acid phosphotyrosine con-taining substrate that simulate residues 988 – 998of the epidermal growth factor receptor(EGFR988–998).Asp48E(-1)A(-3)The table below compares the kinetic parametersof the wild type enzyme with phosphotyrosine pep-tides in which amino acid residues are substituted.L(+1)Тyr46Gln262In the adjacent diagram the structural interactionsbetween active site residues are illustrated for theY(0)first 6 residues of the substrate. Identify which sub-strate residue is most sensitive to substitution andSer216justify your conclusion on the basis of the kineticparameters. Is the decrease in kcat/ KM due to lossof catalytic reactivity or due to suboptimal position-ing of the phospho-Tyr residue in the active site?Phe 182Asp181PTP1ΒSubstratekcatkcat/KmUM10-7 x (s-1 M1)DADEPYLIPQQGDADAPYLIPQGDAAEPYLIPQQGAAAAPYLIPQQG44.6 + 1.839.8 + 0.323.9 + 0.913.7 + 0.461.1 + 0.250.29 + 0.010.53 + 0.0235.3 + 0.226.6 + 0.2234.7 + 0.2552.7 + 0.70.066 + 0.001

Phosphotyrosine protein phosphatases (PTPases): These are a group of enzymes that catalyze the…

(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

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...