(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
9th Edition
ISBN:9781319114671
Author:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
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Chapter1: Biochemistry: An Evolving Science
Section: Chapter Questions
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Question
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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