Effects on general acid catalysis from mutations of the invariant tryptophan and arginine residues in the protein tyrosine phosphatase from Yersinia

Richard H. Hoff, Alvan C. Hengge, Li Wu, Yen Fang Keng, Zhong-Yin Zhang

Research output: Contribution to journalArticle

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Abstract

General acid catalysis in protein tyrosine phosphatases (PTPases) is accomplished by a conserved Asp residue, which is brought into position for catalysis by movement of a flexible loop that occurs upon binding of substrate. With the PTPase from Yersinia, we have examined the effect on general acid catalysis caused by mutations to two conserved residues that are integral to this conformation change. Residue Trp354 is at a hinge of the loop, and Arg409 forms hydrogen bonding and ionic interactions with the phosphoryl group of substrates. Trp354 was mutated to Phe and to Ala, and residue Arg409 was mutated to Lys and to Ala. The four mutant enzymes were studied using steady state kinetics and heavy-atom isotope effects with the substrate p-nitrophenyl phosphate. The data indicate that mutation of the hinge residue Trp354 to Ala completely disables general acid catalysis. In the Phe mutant, general acid catalysis is partially effective, but the proton is only partially transferred in the transition state, in contrast to the native enzyme where proton transfer to the leaving group is virtually complete. Mutation of Arg409 to Lys has a minimal effect on the K(m), while this parameter is increased 30-fold in the Ala mutant. The k(cat) values for R409K and for R409A are about 4 orders of magnitude lower than that for the native enzyme. General acid catalysis is rendered inoperative by the Lys mutation, but partial proton transfer during catalysis still occurs in the Ala mutant. Structural explanations for the differential effects of these mutations on movement of the flexible loop that enables general acid catalysis are presented.

Original languageEnglish (US)
Pages (from-to)46-54
Number of pages9
JournalBiochemistry
Volume39
Issue number1
DOIs
StatePublished - Jan 11 2000
Externally publishedYes

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Yersinia
Protein Tyrosine Phosphatases
Catalysis
Tryptophan
Arginine
Mutation
Acids
Protons
Proton transfer
Hinges
Substrates
Enzymes
Hydrogen Bonding
Viperidae
Isotopes
Conformations
Hydrogen bonds
Atoms
Kinetics

ASJC Scopus subject areas

  • Biochemistry

Cite this

Effects on general acid catalysis from mutations of the invariant tryptophan and arginine residues in the protein tyrosine phosphatase from Yersinia. / Hoff, Richard H.; Hengge, Alvan C.; Wu, Li; Keng, Yen Fang; Zhang, Zhong-Yin.

In: Biochemistry, Vol. 39, No. 1, 11.01.2000, p. 46-54.

Research output: Contribution to journalArticle

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