Abstract
Previous enzyme kinetic and structural studies have revealed a critical role for Asp181 (PTP1B numbering) in PTP (protein-tyrosine phosphatase)-mediated catalysis. In the E-P (phosphoenzyme) formation step, Asp181 functions as a general acid, while in the E-P hydrolysis step it acts as a general base. Most of our understanding of the role of Asp 181 is derived from studies with the Yersinia PTP and the mammalian PTP1B, and to some extent also TC (T-cell)-PTP and the related PTPα and PTPε. The neighbouring residue 182 is a phenylalanine in these four mammalian enzymes and a glutamine in Yersinia PTP. Surprisingly, little attention has been paid to the fact that this residue is a histidine in most other mammalian PTPs. Using a reciprocal single-point mutational approach with introduction of His182 in PTP1B and Phe182 in PTPH1, we demonstrate here that His182-PTPs, in comparison with Phe 182-PTPs, have significantly decreased kcat values, and to a lesser degree, decreased kcat/Km values. Combined enzyme kinetic, X-ray crystallographic and molecular dynamics studies indicate that the effect of His182 is due to interactions with Asp 181 and with Gln262. We conclude that residue 182 can modulate the functionality of both Asp181 and Gln262 and therefore affect the E-P hydrolysis step of PTP-mediated catalysis.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 421-433 |
| Number of pages | 13 |
| Journal | Biochemical Journal |
| Volume | 378 |
| Issue number | 2 |
| DOIs | |
| State | Published - Mar 1 2004 |
| Externally published | Yes |
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Keywords
- Enzyme kinetics
- Molecular dynamics
- Protein-tyrosine phosphatase
- PTP1B
- PTPH1
- X-ray crystallography
ASJC Scopus subject areas
- Biochemistry
Cite this
Residue 182 influences the second step of protein-tyrosine phosphatase-mediated catalysis. / Pedersen, Ana K.; Guo, Xiao Ling; Møller, Karin B.; Peters, Günther H.; Andersen, Henrik S.; Kastrup, Jette S.; Mortensen, Steen B.; Iversen, Lars F.; Zhang, Zhong-Yin; Møller, Niels Peter H.
In: Biochemical Journal, Vol. 378, No. 2, 01.03.2004, p. 421-433.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Residue 182 influences the second step of protein-tyrosine phosphatase-mediated catalysis
AU - Pedersen, Ana K.
AU - Guo, Xiao Ling
AU - Møller, Karin B.
AU - Peters, Günther H.
AU - Andersen, Henrik S.
AU - Kastrup, Jette S.
AU - Mortensen, Steen B.
AU - Iversen, Lars F.
AU - Zhang, Zhong-Yin
AU - Møller, Niels Peter H
PY - 2004/3/1
Y1 - 2004/3/1
N2 - Previous enzyme kinetic and structural studies have revealed a critical role for Asp181 (PTP1B numbering) in PTP (protein-tyrosine phosphatase)-mediated catalysis. In the E-P (phosphoenzyme) formation step, Asp181 functions as a general acid, while in the E-P hydrolysis step it acts as a general base. Most of our understanding of the role of Asp 181 is derived from studies with the Yersinia PTP and the mammalian PTP1B, and to some extent also TC (T-cell)-PTP and the related PTPα and PTPε. The neighbouring residue 182 is a phenylalanine in these four mammalian enzymes and a glutamine in Yersinia PTP. Surprisingly, little attention has been paid to the fact that this residue is a histidine in most other mammalian PTPs. Using a reciprocal single-point mutational approach with introduction of His182 in PTP1B and Phe182 in PTPH1, we demonstrate here that His182-PTPs, in comparison with Phe 182-PTPs, have significantly decreased kcat values, and to a lesser degree, decreased kcat/Km values. Combined enzyme kinetic, X-ray crystallographic and molecular dynamics studies indicate that the effect of His182 is due to interactions with Asp 181 and with Gln262. We conclude that residue 182 can modulate the functionality of both Asp181 and Gln262 and therefore affect the E-P hydrolysis step of PTP-mediated catalysis.
AB - Previous enzyme kinetic and structural studies have revealed a critical role for Asp181 (PTP1B numbering) in PTP (protein-tyrosine phosphatase)-mediated catalysis. In the E-P (phosphoenzyme) formation step, Asp181 functions as a general acid, while in the E-P hydrolysis step it acts as a general base. Most of our understanding of the role of Asp 181 is derived from studies with the Yersinia PTP and the mammalian PTP1B, and to some extent also TC (T-cell)-PTP and the related PTPα and PTPε. The neighbouring residue 182 is a phenylalanine in these four mammalian enzymes and a glutamine in Yersinia PTP. Surprisingly, little attention has been paid to the fact that this residue is a histidine in most other mammalian PTPs. Using a reciprocal single-point mutational approach with introduction of His182 in PTP1B and Phe182 in PTPH1, we demonstrate here that His182-PTPs, in comparison with Phe 182-PTPs, have significantly decreased kcat values, and to a lesser degree, decreased kcat/Km values. Combined enzyme kinetic, X-ray crystallographic and molecular dynamics studies indicate that the effect of His182 is due to interactions with Asp 181 and with Gln262. We conclude that residue 182 can modulate the functionality of both Asp181 and Gln262 and therefore affect the E-P hydrolysis step of PTP-mediated catalysis.
KW - Enzyme kinetics
KW - Molecular dynamics
KW - Protein-tyrosine phosphatase
KW - PTP1B
KW - PTPH1
KW - X-ray crystallography
UR - http://www.scopus.com/inward/record.url?scp=12144290569&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=12144290569&partnerID=8YFLogxK
U2 - 10.1042/BJ20030565
DO - 10.1042/BJ20030565
M3 - Article
C2 - 14572311
AN - SCOPUS:12144290569
VL - 378
SP - 421
EP - 433
JO - Biochemical Journal
JF - Biochemical Journal
SN - 0264-6021
IS - 2
ER -