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The Role of Active Site Residues in ATP Binding and Catalysis in the Methanosarcina thermophila Acetate Kinase.

Ingram-Smith C, Wharton J, Reinholz C, Doucet T, Hesler R, Smith K - Life (Basel) (2015)

Bottom Line: Alteration of Asn211 of PHOSPHATE2, Gly239 of LOOP3, and Gly331 of ADENOSINE greatly reduced catalysis.In particular, Gly331, which is highly conserved throughout the ASKHA superfamily, has the greatest effect on substrate selection.Further investigation into differences between the ATPase domain in MtACK and other acetate kinases that show different substrate preferences will provide us with a better understanding of the diversity of phosphoryl donor selection in this enzyme family.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634, USA. cheryli@clemson.edu.

ABSTRACT
Acetate kinase (ACK), which catalyzes the reversible phosphorylation of acetate by ATP, is a member of the acetate and sugar kinase/heat shock cognate/actin (ASKHA) superfamily. ASKHA family members share a common core fold that includes an ATPase domain with five structural motifs. The PHOSPHATE1 motif has previously been shown to be important for catalysis. We have investigated the role of two of these motifs in the Methanosarcina thermophila ACK (MtACK) and have shown that residues projecting into the ACK active site from the PHOSPHATE2 and ADENOSINE loops and a third highly conserved loop designated here as LOOP3 play key roles in nucleotide triphosphate (NTP) selection and utilization. Alteration of Asn211 of PHOSPHATE2, Gly239 of LOOP3, and Gly331 of ADENOSINE greatly reduced catalysis. In particular, Gly331, which is highly conserved throughout the ASKHA superfamily, has the greatest effect on substrate selection. Alteration at this site strongly skewed MtACK toward utilization of purines over pyrimidines, unlike the wild type enzyme that shows broad NTP utilization. Further investigation into differences between the ATPase domain in MtACK and other acetate kinases that show different substrate preferences will provide us with a better understanding of the diversity of phosphoryl donor selection in this enzyme family.

No MeSH data available.


Related in: MedlinePlus

ACK sequence alignment showing the PHOSPHATE1, PHOSPHATE2, and ADENOSINE motifs as well as LOOP3 and LOOP4 which extend in toward the active site, as shown in Figure 1. Accession numbers: M. thermophila, GI:12084262; E. histolytica, GI:67481281; C. neoformans, GI:427930974; S. typhimurium, GI:16765664; Thermotoga maritima, GI:15643044.
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life-05-00861-f002: ACK sequence alignment showing the PHOSPHATE1, PHOSPHATE2, and ADENOSINE motifs as well as LOOP3 and LOOP4 which extend in toward the active site, as shown in Figure 1. Accession numbers: M. thermophila, GI:12084262; E. histolytica, GI:67481281; C. neoformans, GI:427930974; S. typhimurium, GI:16765664; Thermotoga maritima, GI:15643044.

Mentions: The active site region of the ATPase domain of MtACK (PDB: IG99) in (A) stick and (B) ribbon diagram. Residues shown conform to those in the alignment in Figure 2. ADP is shown in red, PHOSPHATE1 in light blue, PHOSPHATE2 is in light green with Asn211 in dark green, ADENOSINE is in light purple with Gly331 in dark purple, and LOOP3 is in light orange with Gly 239 shown in brown. For clarity, LOOP4 is not shown.


The Role of Active Site Residues in ATP Binding and Catalysis in the Methanosarcina thermophila Acetate Kinase.

Ingram-Smith C, Wharton J, Reinholz C, Doucet T, Hesler R, Smith K - Life (Basel) (2015)

ACK sequence alignment showing the PHOSPHATE1, PHOSPHATE2, and ADENOSINE motifs as well as LOOP3 and LOOP4 which extend in toward the active site, as shown in Figure 1. Accession numbers: M. thermophila, GI:12084262; E. histolytica, GI:67481281; C. neoformans, GI:427930974; S. typhimurium, GI:16765664; Thermotoga maritima, GI:15643044.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4390882&req=5

life-05-00861-f002: ACK sequence alignment showing the PHOSPHATE1, PHOSPHATE2, and ADENOSINE motifs as well as LOOP3 and LOOP4 which extend in toward the active site, as shown in Figure 1. Accession numbers: M. thermophila, GI:12084262; E. histolytica, GI:67481281; C. neoformans, GI:427930974; S. typhimurium, GI:16765664; Thermotoga maritima, GI:15643044.
Mentions: The active site region of the ATPase domain of MtACK (PDB: IG99) in (A) stick and (B) ribbon diagram. Residues shown conform to those in the alignment in Figure 2. ADP is shown in red, PHOSPHATE1 in light blue, PHOSPHATE2 is in light green with Asn211 in dark green, ADENOSINE is in light purple with Gly331 in dark purple, and LOOP3 is in light orange with Gly 239 shown in brown. For clarity, LOOP4 is not shown.

Bottom Line: Alteration of Asn211 of PHOSPHATE2, Gly239 of LOOP3, and Gly331 of ADENOSINE greatly reduced catalysis.In particular, Gly331, which is highly conserved throughout the ASKHA superfamily, has the greatest effect on substrate selection.Further investigation into differences between the ATPase domain in MtACK and other acetate kinases that show different substrate preferences will provide us with a better understanding of the diversity of phosphoryl donor selection in this enzyme family.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634, USA. cheryli@clemson.edu.

ABSTRACT
Acetate kinase (ACK), which catalyzes the reversible phosphorylation of acetate by ATP, is a member of the acetate and sugar kinase/heat shock cognate/actin (ASKHA) superfamily. ASKHA family members share a common core fold that includes an ATPase domain with five structural motifs. The PHOSPHATE1 motif has previously been shown to be important for catalysis. We have investigated the role of two of these motifs in the Methanosarcina thermophila ACK (MtACK) and have shown that residues projecting into the ACK active site from the PHOSPHATE2 and ADENOSINE loops and a third highly conserved loop designated here as LOOP3 play key roles in nucleotide triphosphate (NTP) selection and utilization. Alteration of Asn211 of PHOSPHATE2, Gly239 of LOOP3, and Gly331 of ADENOSINE greatly reduced catalysis. In particular, Gly331, which is highly conserved throughout the ASKHA superfamily, has the greatest effect on substrate selection. Alteration at this site strongly skewed MtACK toward utilization of purines over pyrimidines, unlike the wild type enzyme that shows broad NTP utilization. Further investigation into differences between the ATPase domain in MtACK and other acetate kinases that show different substrate preferences will provide us with a better understanding of the diversity of phosphoryl donor selection in this enzyme family.

No MeSH data available.


Related in: MedlinePlus