Mutation of position 52 in ERK2 creates a nonproductive binding mode for adenosine 5′-triphosphate

Megan J. Robinson, Paul C. Harkins, Jiandong Zhang, Richard Baer, John W. Haycock, Melanie H. Cobb, Elizabeth J. Goldsmith

Research output: Contribution to journalArticle

123 Citations (Scopus)

Abstract

Among the protein kinases, an absolutely conserved lysine in subdomain II is required for high catalytic activity. This lysine is known to interact with the substrate ATP, but otherwise its role is not well understood. We have used biochemical and structural methods to investigate the function of this lysine (K52) in phosphoryl transfer reactions catalyzed by the MAP kinase ERK2. The kinetic properties of activated wild-type ERK2 and K52 mutants were examined using the oncoprotein TAL2, myelin basic protein, and a designed synthetic peptide as substrates. The catalytic activities of K52R and K52A ERK2 were lower than that of wild-type ERK2, primarily as a consequence of reductions in kcat. Further, there was little difference in Km for ATP, but the Km,app for peptide substrate was higher for the K52 mutants. The three-dimensional structure of unphosphorylated K52R ERK2 in the absence and presence of bound ATP was determined and compared with the structure of unphosphorylated wild-type ERK2. ATP adopted a well-defined but distinct binding mode in K52R ERK2 compared to the binding mode in the wild-type enzyme. The structural and kinetic data show that mutation of K52 created a nonproductive binding mode for ATP and suggest that K52 is essential for orienting ATP for catalysis.

Original languageEnglish (US)
Pages (from-to)5641-5646
Number of pages6
JournalBiochemistry
Volume35
Issue number18
StatePublished - May 7 1996

Fingerprint

Adenosine
Adenosine Triphosphate
Mutation
Lysine
Catalyst activity
Substrates
Peptides
Kinetics
Myelin Basic Protein
Oncogene Proteins
Catalysis
Application programs
Protein Kinases
triphosphoric acid
Phosphotransferases
Enzymes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Robinson, M. J., Harkins, P. C., Zhang, J., Baer, R., Haycock, J. W., Cobb, M. H., & Goldsmith, E. J. (1996). Mutation of position 52 in ERK2 creates a nonproductive binding mode for adenosine 5′-triphosphate. Biochemistry, 35(18), 5641-5646.

Mutation of position 52 in ERK2 creates a nonproductive binding mode for adenosine 5′-triphosphate. / Robinson, Megan J.; Harkins, Paul C.; Zhang, Jiandong; Baer, Richard; Haycock, John W.; Cobb, Melanie H.; Goldsmith, Elizabeth J.

In: Biochemistry, Vol. 35, No. 18, 07.05.1996, p. 5641-5646.

Research output: Contribution to journalArticle

Robinson, MJ, Harkins, PC, Zhang, J, Baer, R, Haycock, JW, Cobb, MH & Goldsmith, EJ 1996, 'Mutation of position 52 in ERK2 creates a nonproductive binding mode for adenosine 5′-triphosphate', Biochemistry, vol. 35, no. 18, pp. 5641-5646.
Robinson MJ, Harkins PC, Zhang J, Baer R, Haycock JW, Cobb MH et al. Mutation of position 52 in ERK2 creates a nonproductive binding mode for adenosine 5′-triphosphate. Biochemistry. 1996 May 7;35(18):5641-5646.
Robinson, Megan J. ; Harkins, Paul C. ; Zhang, Jiandong ; Baer, Richard ; Haycock, John W. ; Cobb, Melanie H. ; Goldsmith, Elizabeth J. / Mutation of position 52 in ERK2 creates a nonproductive binding mode for adenosine 5′-triphosphate. In: Biochemistry. 1996 ; Vol. 35, No. 18. pp. 5641-5646.
@article{8a4d4e04e7354dcb8a02e57b90c031dd,
title = "Mutation of position 52 in ERK2 creates a nonproductive binding mode for adenosine 5′-triphosphate",
abstract = "Among the protein kinases, an absolutely conserved lysine in subdomain II is required for high catalytic activity. This lysine is known to interact with the substrate ATP, but otherwise its role is not well understood. We have used biochemical and structural methods to investigate the function of this lysine (K52) in phosphoryl transfer reactions catalyzed by the MAP kinase ERK2. The kinetic properties of activated wild-type ERK2 and K52 mutants were examined using the oncoprotein TAL2, myelin basic protein, and a designed synthetic peptide as substrates. The catalytic activities of K52R and K52A ERK2 were lower than that of wild-type ERK2, primarily as a consequence of reductions in kcat. Further, there was little difference in Km for ATP, but the Km,app for peptide substrate was higher for the K52 mutants. The three-dimensional structure of unphosphorylated K52R ERK2 in the absence and presence of bound ATP was determined and compared with the structure of unphosphorylated wild-type ERK2. ATP adopted a well-defined but distinct binding mode in K52R ERK2 compared to the binding mode in the wild-type enzyme. The structural and kinetic data show that mutation of K52 created a nonproductive binding mode for ATP and suggest that K52 is essential for orienting ATP for catalysis.",
author = "Robinson, {Megan J.} and Harkins, {Paul C.} and Jiandong Zhang and Richard Baer and Haycock, {John W.} and Cobb, {Melanie H.} and Goldsmith, {Elizabeth J.}",
year = "1996",
month = "5",
day = "7",
language = "English (US)",
volume = "35",
pages = "5641--5646",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "18",

}

TY - JOUR

T1 - Mutation of position 52 in ERK2 creates a nonproductive binding mode for adenosine 5′-triphosphate

AU - Robinson, Megan J.

AU - Harkins, Paul C.

AU - Zhang, Jiandong

AU - Baer, Richard

AU - Haycock, John W.

AU - Cobb, Melanie H.

AU - Goldsmith, Elizabeth J.

PY - 1996/5/7

Y1 - 1996/5/7

N2 - Among the protein kinases, an absolutely conserved lysine in subdomain II is required for high catalytic activity. This lysine is known to interact with the substrate ATP, but otherwise its role is not well understood. We have used biochemical and structural methods to investigate the function of this lysine (K52) in phosphoryl transfer reactions catalyzed by the MAP kinase ERK2. The kinetic properties of activated wild-type ERK2 and K52 mutants were examined using the oncoprotein TAL2, myelin basic protein, and a designed synthetic peptide as substrates. The catalytic activities of K52R and K52A ERK2 were lower than that of wild-type ERK2, primarily as a consequence of reductions in kcat. Further, there was little difference in Km for ATP, but the Km,app for peptide substrate was higher for the K52 mutants. The three-dimensional structure of unphosphorylated K52R ERK2 in the absence and presence of bound ATP was determined and compared with the structure of unphosphorylated wild-type ERK2. ATP adopted a well-defined but distinct binding mode in K52R ERK2 compared to the binding mode in the wild-type enzyme. The structural and kinetic data show that mutation of K52 created a nonproductive binding mode for ATP and suggest that K52 is essential for orienting ATP for catalysis.

AB - Among the protein kinases, an absolutely conserved lysine in subdomain II is required for high catalytic activity. This lysine is known to interact with the substrate ATP, but otherwise its role is not well understood. We have used biochemical and structural methods to investigate the function of this lysine (K52) in phosphoryl transfer reactions catalyzed by the MAP kinase ERK2. The kinetic properties of activated wild-type ERK2 and K52 mutants were examined using the oncoprotein TAL2, myelin basic protein, and a designed synthetic peptide as substrates. The catalytic activities of K52R and K52A ERK2 were lower than that of wild-type ERK2, primarily as a consequence of reductions in kcat. Further, there was little difference in Km for ATP, but the Km,app for peptide substrate was higher for the K52 mutants. The three-dimensional structure of unphosphorylated K52R ERK2 in the absence and presence of bound ATP was determined and compared with the structure of unphosphorylated wild-type ERK2. ATP adopted a well-defined but distinct binding mode in K52R ERK2 compared to the binding mode in the wild-type enzyme. The structural and kinetic data show that mutation of K52 created a nonproductive binding mode for ATP and suggest that K52 is essential for orienting ATP for catalysis.

UR - http://www.scopus.com/inward/record.url?scp=0029928988&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029928988&partnerID=8YFLogxK

M3 - Article

C2 - 8639522

AN - SCOPUS:0029928988

VL - 35

SP - 5641

EP - 5646

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 18

ER -