The role of histidines in the acetate kinase from Methanosarcina thermophila.
The role of histidine in the catalytic mechanism of acetate kinase from Methanosarcina thermophila was investigated by diethylpyrocarbonate inactivation and site-directed mutagenesis. Inactivation was accompanied by an increase in absorbance at 240 nm with no change in absorbance at 280 nm, and treatment of the inactivated enzyme with hydroxylamine restored 95% activity, results that indicated diethylpyrocarbonate inactivates the enzyme by the specific modification of histidine. The substrates ATP, ADP, acetate, and acetyl phosphate protected against inactivation suggesting at least one active site where histidine is modified. Correlation of residual activity with the number of histidines modified, as determined by absorbance at 240 nm, indicated that a maximum of three histidines are modified per subunit, two of which are essential for full inactivation. Comparison of the M. thermophila acetate kinase sequence with 56 putative acetate kinase sequences revealed eight highly conserved histidines, three of which (His-123, His-180, and His-208) are perfectly conserved. Diethylpyrocarbonate inactivation of the eight histidine --> alanine variants indicated that His-180 and His-123 are in the active site and that the modification of both is necessary for full inactivation. Kinetic analyses of the eight variants showed that no other histidines are important for activity. Analysis of additional His-180 variants indicated that phosphorylation of His-180 is not essential for catalysis. Possible functions of His-180 are discussed.
Ingram-Smith
C
C
Department of Biochemistry and Molecular Biology, Eberly College of Science, Pennsylvania State University, University Park, Pennsylvania 16802-4500, USA.
Barber
R D
RD
Ferry
J G
JG
D000084:Acetate Kinase / Q000737:chemistry* / Q000502:physiology; D001665:Binding Sites; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D006639:Histidine / Q000502:physiology*; D007700:Kinetics; D017020:Methanosarcina / Q000201:enzymology*; D010766:Phosphorylation; D013329:Structure-Activity Relationship
D006639:Histidine; D000084:Acetate Kinase; D004047:Diethyl Pyrocarbonate