Residue 182 influences the second step of protein-tyrosine phosphatase-mediated catalysis.
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.
Protein Chemistry, Novo Nordisk, DK-2880 Bagsvaerd, Denmark.
Anja K
AK
Pedersen
Xiao-Ling
XL
Guo
Karin B
KB
Møller
Günther H
GH
Peters
Henrik S
HS
Andersen
Jette S
JS
Kastrup
Steen B
SB
Mortensen
Lars F
LF
Iversen
Zhong-Yin
ZY
Zhang
Niels Peter H
NP
Møller
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D002384:Catalysis; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D006639:Histidine / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics; D019000:Phosphotyrosine / Q000378:metabolism; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D054589:Protein Tyrosine Phosphatase, Non-Receptor Type 3; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D014638:Vanadates / Q000737:chemistry
D000596:Amino Acids; D004791:Enzyme Inhibitors; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D010455:Peptides; D019000:Phosphotyrosine; D001224:Aspartic Acid; C008644:nitrophenylphosphate; D014638:Vanadates; D010649:Phenylalanine; D006639:Histidine; C516607:PTPN1 protein, human; C516656:PTPN3 protein, human; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D054589:Protein Tyrosine Phosphatase, Non-Receptor Type 3; D017027:Protein Tyrosine Phosphatases