A direct pyrophosphatase-coupled assay provides new insights into the activation of the secreted adenylate cyclase from Bordetella pertussis by calmodulin.
Continuous recording of the activity of recombinant adenylate cyclase (CyaA) of Bordetella pertussis (EC ) by conductimetric determination of enzyme-coupled pyrophosphate cleavage has enabled us to define a number of novel features of the activation of this enzyme by calmodulin and establish conditions under which valid activation data can be obtained. Activation either in the presence or absence of calcium is characterized by a concentration-dependent lag phase. The rate of formation and breakdown of the activated complex can be determined from an analysis of the lag phase kinetics and is in good agreement with thermodynamic data obtained by measuring the dependence of activation on calmodulin concentration, which show that calcium increases k(on) by about 30-fold. The rate of breakdown of the activated complex, formed either in the presence or absence of calcium, has been determined by dilution experiments and has been shown to be independent of the presence of calcium. The coupled assay is established as a rapid, convenient and safe method which should be readily applicable to the continuous assays of most other enzymes that catalyze reactions in which inorganic pyrophosphate is liberated.
Lawrence
Anthony J
AJ
Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, United Kingdom. A.Lawrence@bio.gla.ac.uk
Coote
John G
JG
Kazi
Yasmin F
YF
Lawrence
Paul D
PD
MacDonald-Fyall
Julia
J
Orr
Barbara M
BM
Parton
Roger
R
Riehle
Mathis
M
Sinclair
James
J
Young
John
J
Price
Nicholas C
NC
D037361:Adenylate Cyclase Toxin; D000262:Adenylyl Cyclases / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001671:Biochemistry / Q000379:methods*; D001886:Bordetella pertussis / Q000201:enzymology*; D002138:Calibration; D002147:Calmodulin / Q000378:metabolism*; D002384:Catalysis; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D011485:Protein Binding; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011755:Pyrophosphatases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013696:Temperature; D013997:Time Factors
D037361:Adenylate Cyclase Toxin; D001426:Bacterial Proteins; D002147:Calmodulin; D011498:Protein Precursors; D011994:Recombinant Proteins; D011755:Pyrophosphatases; D000262:Adenylyl Cyclases