Site-directed mutational analysis of active site residues in the acetate kinase from Methanosarcina thermophila.
Acetate kinase catalyzes the magnesium-dependent transfer of the gamma-phosphate of ATP to acetate. The recently determined crystal structure of the Methanosarcina thermophila enzyme identifies it as a member of the sugar kinase/Hsc70/actin superfamily based on the fold and the presence of five putative nucleotide and metal binding motifs that characterize the superfamily. Residues from four of these motifs in M. thermophila acetate kinase were selected for site-directed replacement and analysis of the variants. Replacement of Asp(148) and Asn(7) resulted in variants with catalytic efficiencies less than 1% of that of the wild-type enzyme, indicating that these residues are essential for activity. Glu(384) was also found to be essential for catalysis. A 30-fold increase in the magnesium concentration required for half-maximal activity of the E384A variant relative to that of the wild type implicated Glu(384) in magnesium binding. The kinetic analysis of variants and structural data is consistent with nonessential roles for active site residues Ser(10), Ser(12), and Lys(14) in catalysis. The results are discussed with respect to the acetate kinase catalytic mechanism and the relationship to other sugar kinase/Hsc70/actin superfamily members.
Miles
R D
RD
Department of Biochemistry, The Pennsylvania State University, University Park, Pennsylvania 16802-4500, USA.
Iyer
P P
PP
Ferry
J G
JG
D000084:Acetate Kinase / Q000737:chemistry* / Q000235:genetics*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D002412:Cations; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D017020:Methanosarcina / Q000201:enzymology* / Q000235:genetics*; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid
D002412:Cations; D000084:Acetate Kinase; D008274:Magnesium