Evidence for a transition state analog, MgADP-aluminum fluoride-acetate, in acetate kinase from Methanosarcina thermophila.
Aluminum fluoride has become an important tool for investigating the mechanism of phosphoryl transfer, an essential reaction that controls a host of vital cell functions. Planar AlF(3) or AlF(4)(-) molecules are proposed to mimic the phosphoryl group in the catalytic transition state. Acetate kinase catalyzes phosphoryl transfer of the ATP gamma-phosphate to acetate. Here we describe the inhibition of acetate kinase from Methanosarcina thermophila by preincubation with MgCl(2), ADP, AlCl(3), NaF, and acetate. Preincubation with butyrate in place of acetate did not significantly inhibit the enzyme. Several NTPs can substitute for ATP in the reaction, and the corresponding NDPs, in conjunction with MgCl(2), AlCl(3), NaF, and acetate, inhibit acetate kinase activity. Fluorescence quenching experiments indicated an increase in binding affinity of acetate kinase for MgADP in the presence of AlCl(3), NaF, and acetate. These and other characteristics of the inhibition indicate that the transition state analog, MgADP-aluminum fluoride-acetate, forms an abortive complex in the active site. The protection from inhibition by a non-hydrolyzable ATP analog or acetylphosphate, in conjunction with the strict dependence of inhibition on the presence of both ADP and acetate, supports a direct in-line mechanism for acetate kinase.
Miles
Rebecca D
RD
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802-4500, USA.
Gorrell
Andrea
A
Ferry
James G
JG
D000084:Acetate Kinase / Q000737:chemistry* / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000737:chemistry* / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D017607:Aluminum Compounds / Q000737:chemistry* / Q000494:pharmacology; D001665:Binding Sites; D002087:Butyrates / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D005459:Fluorides / Q000737:chemistry* / Q000494:pharmacology; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D017020:Methanosarcina / Q000201:enzymology*; D008956:Models, Chemical; D010755:Organophosphates / Q000737:chemistry; D011485:Protein Binding; D012969:Sodium Fluoride / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013997:Time Factors
D017607:Aluminum Compounds; D002087:Butyrates; D004791:Enzyme Inhibitors; D010755:Organophosphates; D015636:Magnesium Chloride; C022571:adenosine 5'-O-(3-thiotriphosphate); C011632:acetyl phosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D012969:Sodium Fluoride; D000084:Acetate Kinase; D005459:Fluorides; C032311:aluminum fluoride