Cold-active citrate synthase: mutagenesis of active-site residues.
A comparison of the crystal structure of the dimeric enzyme citrate synthase from the psychrophilic Arthrobacter strain DS2-3R with that of the structurally homologous enzyme from the hyperthermophilic Pyrococcus furiosus reveals a significant difference in the accessibility of their active sites to substrates. In this work, we investigated the possible role in cold activity of the greater accessibility of the Arthrobacter citrate synthase. By site-directed mutagenesis, we replaced two alanine residues at the entrance to the active site with an arginine and glutamate residue, respectively, as found in the equivalent positions of the Pyrococcus enzyme Also, we introduced a loop into the active site of the psychrophilic citrate synthase, again mimicking the situation in the hyperthermophilic enzyme. Analysis of the thermoactivity and thermostability of the mutant enzymes reveals that cold activity is not significantly compromised by the mutations, but rather the affinity for one of the substrates, acetyl-CoA, is dramatically increased. Moreover, one mutant (Loop insertion/K313L/A361R) has an increased thermostability but a reduced temperature optimum for catalytic activity. This unexpected relationship between stability and activity is discussed with respect to the nature of the dependence of catalytic activity on temperature.
Gerike
U
U
Centre for Extremophile Research, Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK.
Danson
M J
MJ
Hough
D W
DW
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000409:Alanine / Q000737:chemistry / Q000235:genetics; D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D000864:Antarctic Regions; D001173:Arthrobacter / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D002950:Citrate (si)-Synthase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D003080:Cold Temperature; D003461:Crystallography; D019281:Dimerization; D004795:Enzyme Stability / Q000235:genetics; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D020027:Pyrococcus furiosus / Q000201:enzymology / Q000235:genetics; D013329:Structure-Activity Relationship; D013696:Temperature
D018698:Glutamic Acid; D000105:Acetyl Coenzyme A; D002950:Citrate (si)-Synthase; D000409:Alanine