5. Protein tyrosine phosphatase-1B (PTP1B) is an important enzyme regulating insulin signaling be- cause it catalyzes the hydrolysis of phosphorylated tyrosine residues on the insulin receptor and on insulin receptor substrates, proteins of approximately 200,000 molecular weight that serve as cell sig- naling intermediates. The reaction has been shown to adhere to the following mechanism in the scheme below: E + AROPO,2 k3 E-P E + HOPO, 2- E• AROPO, ArОH where ArOPO3²- represents the phosphorylated aromatic group. (a) (. ) With p-nitrophenyl-phosphate (PNPP), a syn- thetic organic substrate under conditions [So] >> Eo], the traces illustrated in the diagram to the right were obtained whereby the optical density at 410 nm monitors the release of the p-nitrophenolate anion (see reaction scheme above) upon cleavage of the ArOPO32- substrate. What is this phe- nomenon called? What information does this observation 0.14 0.12 [PTP1]=0.054 mM 0.1 0.08 0.06 [PTP1]=0.027 mM 0.04 provide about the relative magnitudes of k2 and k3? Why does the amplitude of the initial phase (highlighted in red for the higher enzyme concentration) change with enzyme concentration? What does the slower phase of each trace represent? 0.02 0.02 0.04 0.06 0.08 0.1 Time, (s) :) In steady-state kinetic studies, the following parameters were determined for the synthetic (b) substrate PNPP and for an 11-amino acid peptide containing a phosphorylated tyrosine residue simul- ating part of the epidermal growth factor receptor, a physiologically relevant substrate of the enzyme (EGFR988–998). keat (s) Км (М) kcatd KM (M1 s-1) Substrate p-nitrophenyl- phosphate 43.7 + 4.0 (4.7 + 0.1) х 10 4 9.3 x 104 EGFR988–998 (5.1 ± 1.2) x 10-6 8.7 x 106 44.4 + 4.3 On the basis of these results and your conclusions for part (a) explain why the values Kcat in the table confirm that the rate-limiting step in the reaction is k3, i.e., k2>> k3. What do these results imply about the chemical nature of the E-P reaction intermediate in the equation above for the enzyme catalyzed reaction despite the chemically different substrates for which the kinetic studies were carried out (by an undergraduate student at the UofC). Absorbance at 410 nm
5. Protein tyrosine phosphatase-1B (PTP1B) is an important enzyme regulating insulin signaling be- cause it catalyzes the hydrolysis of phosphorylated tyrosine residues on the insulin receptor and on insulin receptor substrates, proteins of approximately 200,000 molecular weight that serve as cell sig- naling intermediates. The reaction has been shown to adhere to the following mechanism in the scheme below: E + AROPO,2 k3 E-P E + HOPO, 2- E• AROPO, ArОH where ArOPO3²- represents the phosphorylated aromatic group. (a) (. ) With p-nitrophenyl-phosphate (PNPP), a syn- thetic organic substrate under conditions [So] >> Eo], the traces illustrated in the diagram to the right were obtained whereby the optical density at 410 nm monitors the release of the p-nitrophenolate anion (see reaction scheme above) upon cleavage of the ArOPO32- substrate. What is this phe- nomenon called? What information does this observation 0.14 0.12 [PTP1]=0.054 mM 0.1 0.08 0.06 [PTP1]=0.027 mM 0.04 provide about the relative magnitudes of k2 and k3? Why does the amplitude of the initial phase (highlighted in red for the higher enzyme concentration) change with enzyme concentration? What does the slower phase of each trace represent? 0.02 0.02 0.04 0.06 0.08 0.1 Time, (s) :) In steady-state kinetic studies, the following parameters were determined for the synthetic (b) substrate PNPP and for an 11-amino acid peptide containing a phosphorylated tyrosine residue simul- ating part of the epidermal growth factor receptor, a physiologically relevant substrate of the enzyme (EGFR988–998). keat (s) Км (М) kcatd KM (M1 s-1) Substrate p-nitrophenyl- phosphate 43.7 + 4.0 (4.7 + 0.1) х 10 4 9.3 x 104 EGFR988–998 (5.1 ± 1.2) x 10-6 8.7 x 106 44.4 + 4.3 On the basis of these results and your conclusions for part (a) explain why the values Kcat in the table confirm that the rate-limiting step in the reaction is k3, i.e., k2>> k3. What do these results imply about the chemical nature of the E-P reaction intermediate in the equation above for the enzyme catalyzed reaction despite the chemically different substrates for which the kinetic studies were carried out (by an undergraduate student at the UofC). Absorbance at 410 nm
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
Problem 1P
Related questions
Question
![5. Protein tyrosine phosphatase-1B (PTP1B) is an important enzyme regulating insulin signaling be-
cause it catalyzes the hydrolysis of phosphorylated tyrosine residues on the insulin receptor and on
insulin receptor substrates, proteins of approximately 200,000 molecular weight that serve as cell sig-
naling intermediates. The reaction has been shown to adhere to the following mechanism in the
scheme below:
k3
E-P
2-
E + AROPO,
НОРО,*
2-
E •
AROPO,
ArОH
where ArOPO32- represents the phosphorylated aromatic group.
(a) (. ) With p-nitrophenyl-phosphate (PNPP), a syn-
thetic organic substrate under conditions [So] >> Eo], the
traces illustrated in the diagram to the right were obtained
whereby the optical density at 410 nm monitors the release
of the p-nitrophenolate anion (see reaction scheme above)
upon cleavage of the ArOPO32- substrate. What is this phe-
nomenon called? What information does this observation
0.14
0.12
[PTP1]=0.054 mM
0.1
0.08
0.06
[PTP1]=0.027 mM
0.04
provide about the relative magnitudes of k2 and k3? Why
does the amplitude of the initial phase (highlighted in red
for the higher enzyme concentration) change with enzyme
concentration? What does the slower phase of each trace
represent?
0.02
0.02
0.04
0.06
0.08
0.1
Time, (s)
:) In steady-state kinetic studies, the following parameters were determined for the synthetic
(b)
substrate pNPP and for an 11-amino acid peptide containing a phosphorylated tyrosine residue simul-
ating part of the epidermal growth factor receptor, a physiologically relevant substrate of the enzyme
(EGFR988–998).
Substrate
Kcat (s)
Км (М)
Kcat Km (M-1 s-1)
p-nitrophenyl-
phosphate
43.7 + 4.0
(4.7 + 0.1) х 10 4
9.3 x 104
EGFR988–998
(5.1 + 1.2) x 10-6
8.7 x 106
44.4 + 4.3
On the basis of these results and your conclusions for part (a) explain why the values kcat in the table
confirm that the rate-limiting step in the reaction is k3, i.e., k2>> k3. What do these results imply about
the chemical nature of the E-P reaction intermediate in the equation above for the enzyme catalyzed
reaction despite the chemically different substrates for which the kinetic studies were carried out (by
an undergraduate student at the UofC).
Absorbance at 410 nm](/v2/_next/image?url=https%3A%2F%2Fcontent.bartleby.com%2Fqna-images%2Fquestion%2F7e3218aa-1b65-42a2-9071-7edb43c3a555%2F4bfe8c4a-7f80-4223-98b2-91bcfba946cc%2Fftflunj_processed.png&w=3840&q=75)
Transcribed Image Text:5. Protein tyrosine phosphatase-1B (PTP1B) is an important enzyme regulating insulin signaling be-
cause it catalyzes the hydrolysis of phosphorylated tyrosine residues on the insulin receptor and on
insulin receptor substrates, proteins of approximately 200,000 molecular weight that serve as cell sig-
naling intermediates. The reaction has been shown to adhere to the following mechanism in the
scheme below:
k3
E-P
2-
E + AROPO,
НОРО,*
2-
E •
AROPO,
ArОH
where ArOPO32- represents the phosphorylated aromatic group.
(a) (. ) With p-nitrophenyl-phosphate (PNPP), a syn-
thetic organic substrate under conditions [So] >> Eo], the
traces illustrated in the diagram to the right were obtained
whereby the optical density at 410 nm monitors the release
of the p-nitrophenolate anion (see reaction scheme above)
upon cleavage of the ArOPO32- substrate. What is this phe-
nomenon called? What information does this observation
0.14
0.12
[PTP1]=0.054 mM
0.1
0.08
0.06
[PTP1]=0.027 mM
0.04
provide about the relative magnitudes of k2 and k3? Why
does the amplitude of the initial phase (highlighted in red
for the higher enzyme concentration) change with enzyme
concentration? What does the slower phase of each trace
represent?
0.02
0.02
0.04
0.06
0.08
0.1
Time, (s)
:) In steady-state kinetic studies, the following parameters were determined for the synthetic
(b)
substrate pNPP and for an 11-amino acid peptide containing a phosphorylated tyrosine residue simul-
ating part of the epidermal growth factor receptor, a physiologically relevant substrate of the enzyme
(EGFR988–998).
Substrate
Kcat (s)
Км (М)
Kcat Km (M-1 s-1)
p-nitrophenyl-
phosphate
43.7 + 4.0
(4.7 + 0.1) х 10 4
9.3 x 104
EGFR988–998
(5.1 + 1.2) x 10-6
8.7 x 106
44.4 + 4.3
On the basis of these results and your conclusions for part (a) explain why the values kcat in the table
confirm that the rate-limiting step in the reaction is k3, i.e., k2>> k3. What do these results imply about
the chemical nature of the E-P reaction intermediate in the equation above for the enzyme catalyzed
reaction despite the chemically different substrates for which the kinetic studies were carried out (by
an undergraduate student at the UofC).
Absorbance at 410 nm
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