TY - JOUR

T1 - Residue 182 influences the second step of protein-tyrosine phosphatase-mediated catalysis

AU - Pedersen, Anja Kallesøe

AU - Guo, Xiao-Ling

AU - Møller, Karin B

AU - Peters, Günther H

AU - Andersen, Henrik S

AU - Kastrup, Jette Sandholm Jensen

AU - Mortensen, Thomas Steen

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 Asp181 is derived from studies with the Yersinia PTP and the mammalian PTP1B, and to some extent also TC (T-cell)-PTP and the related PTPa and PTPe. 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 Phe182-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 Asp181 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 Asp181 is derived from studies with the Yersinia PTP and the mammalian PTP1B, and to some extent also TC (T-cell)-PTP and the related PTPa and PTPe. 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 Phe182-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 Asp181 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 - Amino Acid Sequence

KW - Amino Acids

KW - Aspartic Acid

KW - Catalysis

KW - Enzyme Inhibitors

KW - Histidine

KW - Humans

KW - Hydrolysis

KW - Models, Chemical

KW - Models, Molecular

KW - Mutagenesis, Site-Directed

KW - Nitrophenols

KW - Organophosphorus Compounds

KW - Peptides

KW - Phenylalanine

KW - Phosphotyrosine

KW - Protein Tyrosine Phosphatase, Non-Receptor Type 1

KW - Protein Tyrosine Phosphatase, Non-Receptor Type 3

KW - Protein Tyrosine Phosphatases

KW - Sequence Alignment

KW - Vanadates

U2 - 10.1042/BJ20030565

DO - 10.1042/BJ20030565

M3 - Journal article

C2 - 14572311

VL - 378

SP - 421

EP - 433

JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

IS - Pt 2

ER -