Structural and kinetic analyses of arginine residues in the active site of the acetate kinase from Methanosarcina thermophila

Andrea Gorrell, Sarah H. Lawrence, James Gregory Ferry

Research output: Contribution to journalArticle

38 Citations (Scopus)

Abstract

Acetate kinase catalyzes transfer of the γ-phosphate of ATP to acetate. The only crystal structure reported for acetate kinase is the homodimeric enzyme from Methanosarcina thermophila containing ADP and sulfate in the active site (Buss, K. A., Cooper, D. C., Ingram-Smith, C., Ferry, J. G., Sanders, D. A., and Hasson, M. S. (2001) J. Bacteriol. 193, 680-686). Here we report two new crystal structure of the M. thermophila enzyme in the presence of substrate and transition state analogs. The enzyme co-crystallized with the ATP analog adenosine 5′-[γ-thio]triphosphate contained AMP adjacent to thiopyrophosphate in the active site cleft of monomer B. The enzyme co-crystallized with ADP, acetate, Al3+, and F- contained a linear array of ADP-AlF3-acetate in the active site cleft of monomer B. Together, the structures clarify the substrate binding sites and support a direct in-line transfer mechanism in which AlF3 mimics the meta-phosphate transition state. Monomers A of both structures contained ADP and sulfate, and the active site clefts were closed less than in monomers B, suggesting that domain movement contributes to catalysis. The finding that His180 was in close proximity to AlF3 is consistent with a role for stabilization of the meta-phosphate that is in agreement with a previous report indicating that this residue is essential for catalysis. Residue Arg241 was also found adjacent to AlF3, consistent with a role for stabilization of the transition state. Kinetic analyses of Arg 241 and Arg91 replacement variants indicated that these residues are essential for catalysis and also indicated a role in binding acetate.

Original languageEnglish (US)
Pages (from-to)10731-10742
Number of pages12
JournalJournal of Biological Chemistry
Volume280
Issue number11
DOIs
StatePublished - Mar 18 2005

Fingerprint

Acetate Kinase
Methanosarcina
Adenosine Diphosphate
Arginine
Catalytic Domain
Acetates
Monomers
Catalysis
Kinetics
Phosphates
Enzymes
Sulfates
Stabilization
Adenosine Triphosphate
Crystal structure
Substrates
Adenosine Monophosphate
Adenosine
Binding Sites

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

@article{aa723da95556494a82408d2da3888df6,
title = "Structural and kinetic analyses of arginine residues in the active site of the acetate kinase from Methanosarcina thermophila",
abstract = "Acetate kinase catalyzes transfer of the γ-phosphate of ATP to acetate. The only crystal structure reported for acetate kinase is the homodimeric enzyme from Methanosarcina thermophila containing ADP and sulfate in the active site (Buss, K. A., Cooper, D. C., Ingram-Smith, C., Ferry, J. G., Sanders, D. A., and Hasson, M. S. (2001) J. Bacteriol. 193, 680-686). Here we report two new crystal structure of the M. thermophila enzyme in the presence of substrate and transition state analogs. The enzyme co-crystallized with the ATP analog adenosine 5′-[γ-thio]triphosphate contained AMP adjacent to thiopyrophosphate in the active site cleft of monomer B. The enzyme co-crystallized with ADP, acetate, Al3+, and F- contained a linear array of ADP-AlF3-acetate in the active site cleft of monomer B. Together, the structures clarify the substrate binding sites and support a direct in-line transfer mechanism in which AlF3 mimics the meta-phosphate transition state. Monomers A of both structures contained ADP and sulfate, and the active site clefts were closed less than in monomers B, suggesting that domain movement contributes to catalysis. The finding that His180 was in close proximity to AlF3 is consistent with a role for stabilization of the meta-phosphate that is in agreement with a previous report indicating that this residue is essential for catalysis. Residue Arg241 was also found adjacent to AlF3, consistent with a role for stabilization of the transition state. Kinetic analyses of Arg 241 and Arg91 replacement variants indicated that these residues are essential for catalysis and also indicated a role in binding acetate.",
author = "Andrea Gorrell and Lawrence, {Sarah H.} and Ferry, {James Gregory}",
year = "2005",
month = "3",
day = "18",
doi = "10.1074/jbc.M412118200",
language = "English (US)",
volume = "280",
pages = "10731--10742",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "11",

}

Structural and kinetic analyses of arginine residues in the active site of the acetate kinase from Methanosarcina thermophila. / Gorrell, Andrea; Lawrence, Sarah H.; Ferry, James Gregory.

In: Journal of Biological Chemistry, Vol. 280, No. 11, 18.03.2005, p. 10731-10742.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Structural and kinetic analyses of arginine residues in the active site of the acetate kinase from Methanosarcina thermophila

AU - Gorrell, Andrea

AU - Lawrence, Sarah H.

AU - Ferry, James Gregory

PY - 2005/3/18

Y1 - 2005/3/18

N2 - Acetate kinase catalyzes transfer of the γ-phosphate of ATP to acetate. The only crystal structure reported for acetate kinase is the homodimeric enzyme from Methanosarcina thermophila containing ADP and sulfate in the active site (Buss, K. A., Cooper, D. C., Ingram-Smith, C., Ferry, J. G., Sanders, D. A., and Hasson, M. S. (2001) J. Bacteriol. 193, 680-686). Here we report two new crystal structure of the M. thermophila enzyme in the presence of substrate and transition state analogs. The enzyme co-crystallized with the ATP analog adenosine 5′-[γ-thio]triphosphate contained AMP adjacent to thiopyrophosphate in the active site cleft of monomer B. The enzyme co-crystallized with ADP, acetate, Al3+, and F- contained a linear array of ADP-AlF3-acetate in the active site cleft of monomer B. Together, the structures clarify the substrate binding sites and support a direct in-line transfer mechanism in which AlF3 mimics the meta-phosphate transition state. Monomers A of both structures contained ADP and sulfate, and the active site clefts were closed less than in monomers B, suggesting that domain movement contributes to catalysis. The finding that His180 was in close proximity to AlF3 is consistent with a role for stabilization of the meta-phosphate that is in agreement with a previous report indicating that this residue is essential for catalysis. Residue Arg241 was also found adjacent to AlF3, consistent with a role for stabilization of the transition state. Kinetic analyses of Arg 241 and Arg91 replacement variants indicated that these residues are essential for catalysis and also indicated a role in binding acetate.

AB - Acetate kinase catalyzes transfer of the γ-phosphate of ATP to acetate. The only crystal structure reported for acetate kinase is the homodimeric enzyme from Methanosarcina thermophila containing ADP and sulfate in the active site (Buss, K. A., Cooper, D. C., Ingram-Smith, C., Ferry, J. G., Sanders, D. A., and Hasson, M. S. (2001) J. Bacteriol. 193, 680-686). Here we report two new crystal structure of the M. thermophila enzyme in the presence of substrate and transition state analogs. The enzyme co-crystallized with the ATP analog adenosine 5′-[γ-thio]triphosphate contained AMP adjacent to thiopyrophosphate in the active site cleft of monomer B. The enzyme co-crystallized with ADP, acetate, Al3+, and F- contained a linear array of ADP-AlF3-acetate in the active site cleft of monomer B. Together, the structures clarify the substrate binding sites and support a direct in-line transfer mechanism in which AlF3 mimics the meta-phosphate transition state. Monomers A of both structures contained ADP and sulfate, and the active site clefts were closed less than in monomers B, suggesting that domain movement contributes to catalysis. The finding that His180 was in close proximity to AlF3 is consistent with a role for stabilization of the meta-phosphate that is in agreement with a previous report indicating that this residue is essential for catalysis. Residue Arg241 was also found adjacent to AlF3, consistent with a role for stabilization of the transition state. Kinetic analyses of Arg 241 and Arg91 replacement variants indicated that these residues are essential for catalysis and also indicated a role in binding acetate.

UR - http://www.scopus.com/inward/record.url?scp=15444365167&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=15444365167&partnerID=8YFLogxK

U2 - 10.1074/jbc.M412118200

DO - 10.1074/jbc.M412118200

M3 - Article

VL - 280

SP - 10731

EP - 10742

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 11

ER -