Does positive charge at the active sites of phosphatases cause a change in mechanism? The effect of the conserved arginine on the transition state for phosphoryl transfer in the protein-tyrosine phosphatase from Yersinia

Richard H. Hoff, Li Wu, Bo Zhou, Zhong-Yin Zhang, Alvan C. Hengge

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Abstract

Positive charge is uniformly present in the active sites of all known phosphatases. The postulate that this charge imparts a change to the mechanism and the transition state for phosphoryl transfer was examined by comparing kinetic isotope effects with the substrate p-nitrophenyl phosphate for reactions of the native protein tyrosine phosphatase from Yersinia with data from mutants in which the conserved arginine residue was mutated to Lys or to Ala. The k(cat) values for both mutants are about 104 less than that of the native enzyme but are still nearly 105-fold faster than the uncatalyzed rate. Steady-state kinetic data as well as isotope effects showed that both mutations interfere with functioning of general acid catalysis. To examine the effect of positive charge on the transition state free of this additional effect, double mutants were made in which general acid catalysis was removed by mutation of Asp356 to either Asn or Ala in addition to the mutation to Arg. The k(cat)/K(m) values of D356A and D356N are 300-360-fold higher than those of R409A/D356A and R409A/D356N suggesting that the side chain of Arg409 contributes 3.4-3.5 kcal/mol to transition-state stabilization. Comparisons of the isotope effects for reactions of the double mutants with data from general acid single mutants show that mutation of Arg to either Lys or to Ala does not significantly affect the transition state for phosphoryl transfer. This indicates that this residue functions to stabilize the transition state but does not alter it from its structure in the uncatalyzed reaction.

Original languageEnglish (US)
Pages (from-to)9514-9521
Number of pages8
JournalJournal of the American Chemical Society
Volume121
Issue number41
DOIs
StatePublished - Oct 20 1999
Externally publishedYes

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Yersinia
Arginine
Protein Tyrosine Phosphatases
Phosphatases
Phosphoric Monoester Hydrolases
Isotopes
Catalytic Domain
Proteins
Mutation
Catalysis
Acids
Kinetics
Phosphates
Enzymes
Stabilization
Substrates

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

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title = "Does positive charge at the active sites of phosphatases cause a change in mechanism? The effect of the conserved arginine on the transition state for phosphoryl transfer in the protein-tyrosine phosphatase from Yersinia",
abstract = "Positive charge is uniformly present in the active sites of all known phosphatases. The postulate that this charge imparts a change to the mechanism and the transition state for phosphoryl transfer was examined by comparing kinetic isotope effects with the substrate p-nitrophenyl phosphate for reactions of the native protein tyrosine phosphatase from Yersinia with data from mutants in which the conserved arginine residue was mutated to Lys or to Ala. The k(cat) values for both mutants are about 104 less than that of the native enzyme but are still nearly 105-fold faster than the uncatalyzed rate. Steady-state kinetic data as well as isotope effects showed that both mutations interfere with functioning of general acid catalysis. To examine the effect of positive charge on the transition state free of this additional effect, double mutants were made in which general acid catalysis was removed by mutation of Asp356 to either Asn or Ala in addition to the mutation to Arg. The k(cat)/K(m) values of D356A and D356N are 300-360-fold higher than those of R409A/D356A and R409A/D356N suggesting that the side chain of Arg409 contributes 3.4-3.5 kcal/mol to transition-state stabilization. Comparisons of the isotope effects for reactions of the double mutants with data from general acid single mutants show that mutation of Arg to either Lys or to Ala does not significantly affect the transition state for phosphoryl transfer. This indicates that this residue functions to stabilize the transition state but does not alter it from its structure in the uncatalyzed reaction.",
author = "Hoff, {Richard H.} and Li Wu and Bo Zhou and Zhong-Yin Zhang and Hengge, {Alvan C.}",
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T1 - Does positive charge at the active sites of phosphatases cause a change in mechanism? The effect of the conserved arginine on the transition state for phosphoryl transfer in the protein-tyrosine phosphatase from Yersinia

AU - Hoff, Richard H.

AU - Wu, Li

AU - Zhou, Bo

AU - Zhang, Zhong-Yin

AU - Hengge, Alvan C.

PY - 1999/10/20

Y1 - 1999/10/20

N2 - Positive charge is uniformly present in the active sites of all known phosphatases. The postulate that this charge imparts a change to the mechanism and the transition state for phosphoryl transfer was examined by comparing kinetic isotope effects with the substrate p-nitrophenyl phosphate for reactions of the native protein tyrosine phosphatase from Yersinia with data from mutants in which the conserved arginine residue was mutated to Lys or to Ala. The k(cat) values for both mutants are about 104 less than that of the native enzyme but are still nearly 105-fold faster than the uncatalyzed rate. Steady-state kinetic data as well as isotope effects showed that both mutations interfere with functioning of general acid catalysis. To examine the effect of positive charge on the transition state free of this additional effect, double mutants were made in which general acid catalysis was removed by mutation of Asp356 to either Asn or Ala in addition to the mutation to Arg. The k(cat)/K(m) values of D356A and D356N are 300-360-fold higher than those of R409A/D356A and R409A/D356N suggesting that the side chain of Arg409 contributes 3.4-3.5 kcal/mol to transition-state stabilization. Comparisons of the isotope effects for reactions of the double mutants with data from general acid single mutants show that mutation of Arg to either Lys or to Ala does not significantly affect the transition state for phosphoryl transfer. This indicates that this residue functions to stabilize the transition state but does not alter it from its structure in the uncatalyzed reaction.

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